Systems and methods for high-resolution imaging

ABSTRACT

Methods of imaging described herein comprises disposing a first and second ultrasound-switchable fluorophore in an environment; exposing the environment to an ultrasound beam to create an activation region; disposing the first and/or second fluorophore within the activation region to switch the first and/or second fluorophore from an off state to an on state; exposing the environment to a beam of electromagnetic radiation; detecting a first photoluminescence signal at a first location within the environment, the photoluminescence signal comprising at least one of a first ultrasound fluorescence signal emitted by the first fluorophore, a first fluorescence signal emitted by the second fluorophore, and a background signal; correlating the first photoluminescence signal with a first reference signal to generate a correlation coefficient for the first location; and multiplying the first photoluminescence signal by the first correlation coefficient for the first location to generate a first modified photoluminescence signal for the first location.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/615,993, filed Feb. 6, 2015, which is a continuation-in-part of U.S.patent application Ser. No. 14/162,375, filed Jan. 23, 2014, whichclaims priority pursuant to 35 U.S.C. § 119(e) to U.S. ProvisionalPatent Application Ser. No. 61/756,065, filed on Jan. 24, 2013, each ofwhich is hereby incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under contract7R15EB012312-02 awarded by the National Institutes of Health through theNational Institute of Biomedical Imaging and Bioengineering, contractRP120052 awarded by the Cancer Prevention and Research Institute ofTexas, and contract CBET-1253199 awarded by the National ScienceFoundation. The government has certain rights in the invention.

FIELD

This invention relates to systems and methods for high-resolutionimaging and, in particular, to imaging using ultrasound-switchablefluorescence (USF).

BACKGROUND

Fluorescence imaging in deep biological tissue can provide importantinformation regarding tissue structure, function, and dysfunction.However, some previous fluorescence imaging techniques are limited inpenetration depth and/or spatial resolution due to strong lightscattering in deep tissue. Some previous fluorescence imaging techniquesare also limited in signal-to-noise ratio (SNR). As a result, suchmethods can have reduced effectiveness for many tissue biology and/orclinical applications.

Therefore, there exists a need for improved systems and methods forhigh-resolution imaging, particularly for imaging deep biologicaltissue.

SUMMARY

In one aspect, methods of imaging are described herein which, in somecases, can provide one or more advantages compared to other methods. Forexample, in some embodiments, a method described herein can provideimaging of deep biological tissue with a resolution beyond the acousticdiffraction limit and can further exhibit an improved imagingdepth-to-imaging-resolution ratio (DRR) and/or an improvedsignal-to-noise ratio (SNR). In addition, a fluorophore of a methoddescribed herein, in some cases, can exhibit a large on/off ratio offluorescence intensity or lifetime and/or a narrow transition bandwidthbetween on and off states. Further, a fluorophore of a method describedherein can also exhibit a tunable threshold between on and off states.Moreover, in some instances, a method described herein can permitmultiplexed ultrasound fluorescence imaging, including to simultaneouslyimage multiple molecular targets, such as may be desirable for one ormore biomedical applications.

A method described herein, in some embodiments, comprises disposing apopulation of ultrasound-switchable fluorophores in a biologicalenvironment, the fluorophores having a switching threshold between anoff state and an on state, and exposing the biological environment to anultrasound beam to create an activation region within the biologicalenvironment. The method further comprises switching at least one of thefluorophores within the activation region from the off state to the onstate, exciting the at least one fluorophore with a beam ofelectromagnetic radiation, and detecting light emitted by the at leastone fluorophore. In some embodiments, the activation region has amaximum negative pressure and/or a maximum temperature, and theswitching threshold of the at least one fluorophore is at least about 50percent of the maximum negative pressure of the activation region or atleast about 50 percent of the maximum temperature of the activationregion. In some cases, for instance, the fluorophores have a switchingtemperature threshold between an off state and an on state, and theactivation region has a maximum temperature that is greater than orequal to the switching temperature threshold of the fluorophores.

In addition, in some embodiments, a method described herein comprisesexposing the biological environment to a plurality of ultrasound beamsfrom a plurality of different directions, wherein the focal zones of theultrasound beams at least partially overlap. Further, in some suchcases, the switching threshold of the fluorophores is greater than themaximum temperature or maximum negative pressure provided by the focalzone of one of the ultrasound beams alone.

Moreover, in some cases, a method described herein further comprisesexposing the biological environment to a pulsed beam of electromagneticradiation prior to exposing the biological environment to the ultrasoundbeam, the pulsed beam having a pulse duration of no greater than 100picoseconds (ps), wherein the pulse duration is defined as the fullwidth at half maximum of the optical power of the pulse over time.

Additionally, in some instances, a method described herein comprises (a)disposing a first ultrasound-switchable fluorophore in an environment,such as a biological environment, (b) exposing the environment to anultrasound beam to create an activation region within the environment,(c) disposing the first fluorophore within the activation region toswitch the first fluorophore from an off state to an on state, and (d)exposing the environment to a beam of electromagnetic radiation, therebyexciting the first fluorophore. In some cases, exposing the environmentto the beam of electromagnetic radiation also excites at least onephotoluminescent background species present in the environment.Moreover, a method described can also include (e) detecting a firstphotoluminescence signal at a first location within the environment, thefirst photoluminescence signal comprising at least one of a firstultrasound fluorescence signal emitted by the first fluorophore and afirst background signal. In some instances, the first background signalcomprises a first background photoluminescence signal emitted by the atleast one photoluminescent background species. In addition, in someembodiments, the method further comprises (f) correlating the firstphotoluminescence signal with a first reference signal to generate afirst correlation coefficient for the first location and (g) multiplyingthe first photoluminescence signal by the correlation coefficient forthe first location to generate a first modified photoluminescence signalfor the first location. The first reference signal of a method describedherein, in some cases, corresponds to the first ultrasound fluorescencesignal of the first fluorophore. Further, in some cases, correlating thefirst photoluminescence signal with the first reference signal comprisescomparing a temporal intensity decay profile of the firstphotoluminescence signal to a temporal intensity decay profile of thefirst reference signal.

In addition, the foregoing steps (e)-(g) of detecting and processing aphotoluminescence signal at a first location within the environment canbe repeated any desired number of times to generate a plurality ofmodified photoluminescence signals for a plurality of locations withinthe environment. For example, in some cases, a method described hereinfurther comprises (e₂) detecting a second photoluminescence signal at asecond location within the environment, the second photoluminescencesignal comprising at least one of a second ultrasound fluorescencesignal emitted by the first fluorophore and a second background signal,(f₂) correlating the second photoluminescence signal with the firstreference signal to generate a correlation coefficient for the secondlocation, and (g₂) multiplying the second photoluminescence signal bythe correlation coefficient for the second location to generate a secondmodified photoluminescence signal for the second location. Moregenerally, n modified photoluminescence signals can be generated from nphotoluminescence signals at n locations within the environment and fromn correlation coefficients for the n locations, wherein n can be anydesired integer, such as an integer between 2 and 1000. In this manner,a spatial plot or profile of ultrasound fluorescence emitted by thefirst fluorophore within the environment can be obtained, as describedfurther hereinbelow.

Further, in some cases, a method described herein can include thesimultaneous use of more than one ultrasound-switchable fluorophore.Thus, a method described herein, in some instances, can permit orprovide multiplexed ultrasound fluorescence imaging, includingmultiplexed imaging using a plurality of differing ultrasound-switchablefluorophores.

Moreover, in embodiments, a method of multiplexed imaging describedherein comprises (a) disposing a first ultrasound-switchable fluorophoreand a second ultrasound-switchable fluorophore in an environment, (b)exposing the environment to an ultrasound beam to create an activationregion within the environment, (c) disposing the first fluorophorewithin the activation region to switch the first fluorophore from an offstate to an on state and/or disposing the second fluorophore within theactivation region to switch the second fluorophore from an off state toan on state, (d) exposing the environment to a beam of electromagneticradiation, thereby exciting the first fluorophore and/or the secondfluorophore, (e) detecting a first photoluminescence signal at a firstlocation within the environment, the first photoluminescence signalcomprising at least one of a first ultrasound fluorescence signalemitted by the first fluorophore and a first ultrasound fluorescencesignal emitted by the second fluorophore, and (f) orthogonallydecomposing the first photoluminescence signal into a first basis vectorcorresponding to a normalized ultrasound fluorescence signal of thefirst fluorophore and a second basis vector corresponding to anormalized ultrasound fluorescence signal of the second fluorophore.Additionally, in some cases, a method described herein further comprises(g₁) determining a basis vector coefficient a for the normalizedultrasound fluorescence signal of the first fluorophore at the firstlocation and (g₂) determining a basis vector coefficient b for thenormalized ultrasound fluorescence signal of the second fluorophore atthe first location. A method described herein may also comprise (h₁)multiplying the normalized ultrasound fluorescence signal of the firstfluorophore by the coefficient a to generate a separated ultrasoundfluorescence signal of the first fluorophore at the first location, and(h₂) multiplying the normalized ultrasound fluorescence signal of thesecond fluorophore by the coefficient b to generate a separatedultrasound fluorescence signal of the second fluorophore at the firstlocation. Moreover, as described further hereinbelow, the forgoingprocess of steps (e)-(h) can be repeated any desired number of times togenerate separated ultrasound fluorescence signals of the first and/orsecond fluorophores at any desired number of additional locations withinthe environment.

Additionally, it is also possible to generate separate spatial plots ofultrasound fluorescence emitted by the first and second fluorophoreswithin the environment, including by (i₁) combining the separatedultrasound fluorescence signal of the first fluorophore at the firstlocation with n additional separated ultrasound fluorescence signals ofthe first fluorophore at n additional locations to generate a spatialplot of ultrasound fluorescence emitted by the first fluorophore withinthe environment, and (i₂) combining the separated ultrasoundfluorescence signal of the second fluorophore at the first location withn additional separated ultrasound fluorescence signals of the secondfluorophore at n additional locations to generate a spatial plot ofultrasound fluorescence emitted by the second fluorophore within theenvironment. Moreover, once separated ultrasound fluorescence signals ofthe first and second fluorophores are obtained for one or more locationswithin the environment, it is possible, if desired, to improve the SNRof these signals in a manner described hereinabove.

In some embodiments, an ultrasound-switchable fluorophore used in amethod described herein comprises a Förster resonance energy transfer(FRET) donor species and a FRET acceptor species. In some suchinstances, the distance between the FRET donor species and the FRETacceptor species of the fluorophore is altered by the presence of anultrasound beam. For example, in some embodiments, a fluorophorecomprises a microbubble comprising one or more FRET donor species andone or more FRET acceptor species attached to the surface of themicrobubble. In other cases, an ultrasound-switchable fluorophore usedin a method described herein comprises a thermoresponsive polymer.Additionally, in some cases, a thermoresponsive polymer of a fluorophoredescribed herein comprises one or more fluorescent moieties or isconjugated to one or more fluorescent species, such as one or morefluorescent dye molecules. In other instances, a fluorophore describedherein comprises a fluorescent material dispersed in and/or attached tothe surface of a thermoresponsive polymer nanoparticle. Moreover, insome embodiments, an ultrasound-switchable fluorophore described hereinexhibits a fluorescence emission profile in the near infrared (NIR), anon-to-off ratio in fluorescence intensity (I_(On)/I_(Off)) of at leastabout 2, an on-to-off ratio in fluorescence lifetime (τ_(On)/τ_(Off)) ofat least about 1.5, and/or a transition bandwidth between on and offstates (T_(BW)) of no greater than about 10° C.

In addition, in some embodiments, the biological environment of a methoddescribed herein comprises deep tissue. In some cases, the biologicalenvironment comprises tumor vasculature. Moreover, in some instances, amethod described herein exhibits a penetration depth/resolution ratio ofat least about 100.

These and other embodiments are described in more detail in the detaileddescription which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an ultrasound-switchable fluorescence processaccording to one embodiment of a method described herein.

FIG. 2 illustrates an ultrasound-switchable fluorescence processaccording to one embodiment of a method described herein.

FIG. 3 illustrates plots of the fluorescence intensity and fluorescencelifetime of a temperature-dependent fluorophore suitable for use in someembodiments of methods described herein.

FIG. 4 illustrates the structures of components of fluorophores suitablefor use in some embodiments of methods described herein.

FIG. 5 illustrates fluorescence switching curves of fluorophoressuitable for use in some embodiments of methods described herein.

FIG. 6 illustrates fluorescence data for a fluorophore suitable for usein some embodiments of methods described herein.

FIG. 7 illustrates a system used to measure the fluorescencecharacteristics of fluorophores suitable for use in some embodiments ofmethods described herein.

FIG. 8A illustrates components and steps of a method of imagingaccording to one embodiment described herein. FIG. 8B illustratescomponents and steps of a method of imaging according to one embodimentdescribed herein. FIG. 8C illustrates steps of a method of imagingaccording to one embodiment described herein.

FIG. 9A illustrates a USF image obtained by a method according to oneembodiment described herein. FIG. 9B illustrates a comparative imagecorresponding to the image of FIG. 9A. FIG. 9C illustrates afluorescence profile obtained by a method according to one embodimentdescribed herein. FIG. 9D illustrates a fluorescence profile obtained bya method according to one embodiment described herein.

FIG. 10 illustrates an ultrasound-switchable fluorescence processaccording to one embodiment of a method described herein.

FIG. 11 illustrates an ultrasound-switchable fluorescence processaccording to one embodiment of a method described herein.

FIG. 12 illustrates an ultrasound-switchable fluorescence processaccording to one embodiment of a method described herein.

FIG. 13 illustrates the structure of a component of a fluorophoresuitable for use in some embodiments of methods described herein.

FIG. 14 illustrates a plot of imaging resolution versus fluorophoreswitching threshold for some embodiments of methods described herein.

FIG. 15 illustrates an ultrasound-switchable fluorescence processaccording to one embodiment of a method described herein.

FIG. 16 illustrates an ultrasound-switchable fluorescence processaccording to one embodiment of a method described herein.

FIG. 17 illustrates an ultrasound-switchable fluorescence processaccording to one embodiment of a method described herein.

FIG. 18A and FIG. 18B each illustrates the structure of fluorescentmaterials suitable for use in ultrasound-switchable fluorophoresaccording to some embodiments described herein.

FIG. 19A-19F each illustrates emission profiles of ultrasound-switchablefluorophores according to some embodiments described herein.

FIG. 20A and FIG. 20B each illustrates emission profiles ofultrasound-switchable fluorophores according to some embodimentsdescribed herein.

FIG. 21A illustrates components and steps of a method of imagingaccording to one embodiment described herein. FIG. 21B illustrates stepsof a method of imaging corresponding to the components of FIG. 21A. FIG.21C illustrates components and steps of a method of imaging according toone embodiment described herein. FIG. 21D illustrates components andsteps of a method of imaging according to one embodiment describedherein. FIG. 21E illustrates steps of methods of imaging correspondingto the components of FIG. 21C and FIG. 21D. FIG. 21F illustratescomponents and steps of a method of imaging according to one embodimentdescribed herein. FIG. 21G illustrates steps of a method of imagingcorresponding to the components of FIG. 21F.

FIG. 22A illustrates the emission profile of an ultrasound-switchablefluorophore according to one embodiment described herein. FIG. 22Billustrates the emission profile of a background signal associated withthe fluorophore of FIG. 22A. FIG. 22C and FIG. 22D each illustrate anultrasound fluorescence emission profile for the fluorophore of FIG.22A. FIG. 22E illustrates the emission profile of anultrasound-switchable fluorophore according to one embodiment describedherein. FIG. 22F illustrates the emission profile of a background signalassociated with the fluorophore of FIG. 22E. FIG. 22G and FIG. 2211 eachillustrate an ultrasound fluorescence emission profile for thefluorophore of FIG. 22E.

FIGS. 23A-23D each illustrate an ultrasound fluorescence emissionprofile of an ultrasound-switchable fluorophore according to someembodiments described herein.

FIG. 24A and FIG. 24B each illustrates an ultrasound fluorescenceemission profile of an ultrasound-switchable fluorophore according toone embodiment described herein. FIG. 24C illustrates a totalphotoluminescence signal detected according to one embodiment describedherein.

DETAILED DESCRIPTION

Embodiments described herein can be understood more readily by referenceto the following detailed description, examples, and figures. Elements,apparatus, and methods described herein, however, are not limited to thespecific embodiments presented in the detailed description, examples,and figures. It should be recognized that these embodiments are merelyillustrative of the principles of the present invention. Numerousmodifications and adaptations will be readily apparent to those of skillin the art without departing from the spirit and scope of the invention.

In addition, all ranges disclosed herein are to be understood toencompass any and all subranges subsumed therein. For example, a statedrange of “1.0 to 10.0” should be considered to include any and allsubranges beginning with a minimum value of 1.0 or more and ending witha maximum value of 10.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or3.6 to 7.9.

All ranges disclosed herein are also to be considered to include the endpoints of the range, unless expressly stated otherwise. For example, arange of “between 5 and 10” should generally be considered to includethe end points 5 and 10.

Further, when the phrase “up to” is used in connection with an amount orquantity, it is to be understood that the amount is at least adetectable amount or quantity. For example, a material present in anamount “up to” a specified amount can be present from a detectableamount and up to and including the specified amount.

In one aspect, methods of imaging are described herein. In someembodiments, a method of imaging comprises disposing a population ofultrasound-switchable fluorophores in a biological environment, thefluorophores having a switching threshold between an off state and an onstate; and exposing the biological environment to an ultrasound beam tocreate an activation region within the biological environment, theactivation region having a maximum negative pressure and a maximumtemperature, wherein the switching threshold of the at least onefluorophore is at least about 50 percent of the maximum negativepressure or at least about 50 percent of the maximum temperature of theactivation region. The method further comprises switching at least oneof the fluorophores within the activation region from the off state tothe on state; exciting the at least one fluorophore with a beam ofelectromagnetic radiation; and detecting light emitted by the at leastone fluorophore. In some embodiments, the switching threshold of the atleast one fluorophore is at least about 60 percent or at least about 70percent of the maximum negative pressure or maximum temperature of theactivation region. In some cases, the switching threshold of the atleast one fluorophore is between about 60 percent and about 100 percent,between about 60 percent and about 90 percent, between about 70 percentand about 100 percent, between about 70 percent and about 95 percent, orbetween about 70 percent and about 90 percent of the maximum negativepressure or maximum temperature of the activation region. As describedfurther hereinbelow, selecting such a switching threshold, in somecases, can permit improved imaging resolution by effectively reducingthe volume of the activation region to a size below the size of thefocal zone of the ultrasonic beam used to form the activation region.

In other embodiments, a method of imaging comprises disposing apopulation of ultrasound-switchable fluorophores in a biologicalenvironment, the fluorophores having a switching threshold between anoff state and an on state; and exposing the biological environment to aplurality of ultrasound beams from a plurality of different directionsto create an activation region within the biological environment, thefocal zones of the ultrasound beams at least partially overlapping. Insome cases, for example, two orthogonal ultrasound beams are used.Additionally, in some instances, the switching threshold of thefluorophores is greater than the maximum negative pressure or themaximum temperature provided by the focal zone of one of the ultrasoundbeams alone. The method further comprises switching at least one of thefluorophores within the activation region from the off state to the onstate; exciting the at least one fluorophore with a beam ofelectromagnetic radiation; and detecting light emitted by the at leastone fluorophore. As described further hereinbelow, the use of multipleultrasound beams in a manner described herein can permit improvedimaging resolution by reducing the size of the activation region.

In still other embodiments, a method of imaging comprises disposing apopulation of ultrasound-switchable fluorophores in a biologicalenvironment, the fluorophores having a switching threshold between anoff state and an on state; and exposing the biological environment to apulsed beam of electromagnetic radiation, the pulsed beam having a pulseduration of no greater than 100 ps, based on the FWHM of the opticalpower of the pulsed beam over time. The method further comprisesexposing the biological environment to an ultrasound beam to create anactivation region within the biological environment; switching at leastone of the fluorophores within the activation region from the off stateto the on state; exciting the at least one fluorophore with a secondbeam of electromagnetic radiation; and detecting light emitted by the atleast one fluorophore. Moreover, in some cases, the pulsed beam has apulse duration of no greater than about 50 ps or no greater than about10 ps. In some embodiments, the pulsed beam has a pulse duration betweenabout 1 ps and about 100 ps, between about 1 ps and about 10 ps, betweenabout 1 ps and about 50 ps, between about 10 ps and about 100 ps, orbetween about 10 ps and about 50 ps. As described further hereinbelow,exposing the biological environment to a pulsed beam of electromagneticradiation in a manner described herein, in some embodiments, can improvethe signal-to-noise ratio (SNR) of the method by permitting temporalseparation of the detection of the fluorophore emission, compared to theemission from other species present in the biological environment. Thus,in some cases, the step of detecting light emitted by the at least onefluorophore is carried out after a delay corresponding to thefluorescence lifetime of such other species. In addition, in someembodiments, the wavelength of the pulsed beam is selected tosubstantially overlap with the absorption profile of one or more speciespresent in the biological environment.

In yet other embodiments, a method of imaging comprises (a) disposing afirst ultrasound-switchable fluorophore or a first population ofultrasound-switchable fluorophores in an environment, such as abiological environment, (b) exposing the environment to an ultrasoundbeam to create an activation region within the environment, (c)disposing the first fluorophore within the activation region to switchthe first fluorophore from an off state to an on state, and (d) exposingthe environment to a beam of electromagnetic radiation, thereby excitingthe first fluorophore. In some cases, exposing the environment to thebeam of electromagnetic radiation also excites at least onephotoluminescent background species present in the environment.Moreover, the method further comprises (e) detecting a firstphotoluminescence signal at a first location within the environment, thefirst photoluminescence signal comprising at least one of a firstultrasound fluorescence signal emitted by the first fluorophore and afirst background signal. In some instances, the first background signalcomprises a first background photoluminescence signal emitted by atleast one photoluminescent background species. Further, in someembodiments, a method described herein also comprises (f) correlatingthe first photoluminescence signal with a first reference signal togenerate a first correlation coefficient for the first location. In somecases, the first reference signal corresponds to the first ultrasoundfluorescence signal of the first fluorophore. In addition, a methoddescribed herein can also include (g) multiplying the firstphotoluminescence signal by the correlation coefficient for the firstlocation to generate a first modified photoluminescence signal for thefirst location.

Moreover, it is to be understood that the foregoing steps (e)-(g) ofdetecting and processing a photoluminescence signal at a first locationwithin the environment can be repeated any desired number of times togenerate a plurality of modified photoluminescence signals for aplurality of locations within the environment. For example, in somecases, a method described herein further comprises (e₂) detecting asecond photoluminescence signal at a second location within theenvironment, the second photoluminescence signal comprising at least oneof a second ultrasound fluorescence signal emitted by the firstfluorophore and a second background signal, (f₂) correlating the secondphotoluminescence signal with the first reference signal to generate acorrelation coefficient for the second location, and (g₂) multiplyingthe second photoluminescence signal by the correlation coefficient forthe second location to generate a second modified photoluminescencesignal for the second location.

Similarly, the same process of steps (e)-(g) or (e₂)-(g₂) may berepeated to generate a third modified photoluminescence signal from athird photoluminescence signal at a third location within theenvironment and a correlation coefficient for the third location. Moregenerally, n modified photoluminescence signals can be generated from nphotoluminescence signals at n locations within the environment and fromn correlation coefficients for the n locations, wherein n can be anydesired integer, such as an integer between 2 and 1000, between 2 and500, between 2 and 100, between 2 and 50, between 2 and 30, or between 2and 20. In some cases, n may be greater than 1000. Additionally, thenlocations can be any set of desired locations within the environment,including locations that are contiguous or non-contiguous in one or moredirections within the environment. Moreover, a location within anenvironment described herein, in some embodiments, can be a voxel withinthe environment and/or a location identified by one or more of anx-coordinate, a y-coordinate, and a z-coordinate. In some cases, forinstance, a location is a voxel centered at an xy-coordinate value orordered pair (x, y) of the environment or an xyz-coordinate value (x, y,z), such as may be identified for a raster scan of locations within theenvironment. In this manner, a spatial plot or profile of ultrasoundfluorescence emitted by the first fluorophore within the environment canbe obtained. Additionally, as described further hereinbelow, such a plotcan have an improved SNR, including compared to an otherwise similarplot generated without carrying out the correlating and multiplyingsteps described hereinabove.

Thus, in some embodiments, a method of imaging described herein furthercomprises (e_(n)) detecting n additional photoluminescence signals at nadditional locations within the environment, the n additionalphotoluminescence signals comprising at least one of an nth additionalultrasound fluorescence signal emitted by the first fluorophore and annth additional background signal, (f_(n)) correlating the n additionalphotoluminescence signals with the first reference signal to generate nadditional correlation coefficients for the n additional locations, and(g_(n)) multiplying the n additional photoluminescence signals by the nadditional correlation coefficients to generate n additional modifiedphotoluminescence signals for the n additional locations, wherein n isan integer between 1 and 1000. Moreover, such a method can also comprise(h) combining the first modified photoluminescence signal for the firstlocation, the second modified photoluminescence signal for the secondlocation, and the n additional modified photoluminescent signals for then additional locations to generate a spatial plot of ultrasoundfluorescence emitted by the first fluorophore within the environment.

As described further hereinbelow, generating a spatial plot ofultrasound fluorescence emitted by an ultrasound-switchable fluorophorewithin an environment in a manner described herein, in some embodiments,can improve the SNR of the method by reducing the intensity of anybackground photoluminescence that may be present, such as backgroundfluorescence emitted by a fluorescent background species. It is to beunderstood that a “background” species, for reference purposes herein,is a species other than an ultrasound-switchable fluorophore present inthe environment. More particularly, a background species can be aspecies that is not an imaging analyte of the method. In some instances,a background species can comprise, consist, or consist essentially ofbiological tissue present in an imaged biological environment, such thatthe background signal comprises, consists, or consists essentially oftissue autofluorescence.

In addition, in some cases, a method described herein can include thesimultaneous use of more than one ultrasound-switchable fluorophore.Thus, a method described herein, in some instances, can permit orprovide multiplexed ultrasound fluorescence imaging, includingmultiplexed imaging using a plurality of differing ultrasound-switchablefluorophores. For example, in some embodiments, a method describedherein comprises (a) disposing a first ultrasound-switchable fluorophoreand a second ultrasound-switchable fluorophore in an environment, (b)exposing the environment to an ultrasound beam to create an activationregion within the environment, (c) disposing the first fluorophorewithin the activation region to switch the first fluorophore from an offstate to an on state and/or disposing the second fluorophore within theactivation region to switch the second fluorophore from an off state toan on state, (d) exposing the environment to a beam of electromagneticradiation, thereby exciting the first fluorophore and/or the secondfluorophore, and (e) detecting a first photoluminescence signal at afirst location within the environment. The first photoluminescencesignal can comprise at least one of a first ultrasound fluorescencesignal emitted by the first fluorophore, a first fluorescence signalemitted by the second fluorophore, and a first background signal.Moreover, the method can further comprise (f) correlating the firstphotoluminescence signal with a first reference signal to generate afirst correlation coefficient for the first location and (g) multiplyingthe first photoluminescence signal by the first correlation coefficientfor the first location to generate a first modified photoluminescencesignal for the first location.

Moreover, in such a method, the detection, correlation, and other signalprocessing steps (e)-(g) can be carried out in the same manner and/orrepeated as described herein for embodiments using only oneultrasound-switchable fluorophore or population of ultrasound-switchablefluorophores. For example, in some cases, a correlation coefficient isdetermined in a manner described herein for one ultrasound-switchablefluorophore. In addition, in some embodiments, a method described hereinfurther comprises (e_(n)) detecting n additional photoluminescencesignals at n additional locations within the environment, wherein the nadditional photoluminescence signals comprising at least one of an nthadditional ultrasound fluorescence signal emitted by the firstfluorophore, an nth additional ultrasound fluorescence signal emitted bythe second fluorophore, and an nth additional background signal. Such amethod may also comprise the step of (f_(n)) correlating the nadditional photoluminescence signals with the first reference signal togenerate n additional first correlation coefficients for the nadditional locations and (g_(n)) multiplying the n additionalphotoluminescence signals by the n additional first correlationcoefficients to generate n additional modified photoluminescence signalsfor the n additional locations, wherein n is an integer between 1 and1000.

Thus, as described above for embodiments using one ultrasound-switchablefluorophore, a method described herein can be used to generate a spatialprofile or plot of ultrasound fluorescence emitted by anultrasound-switchable fluorophore present in the environment. Moreover,it is to be understood that the fluorophore associated with a specificspatial plot can be determined by the choice of reference signal. Forexample, in some cases, the first reference signal corresponds to thefirst ultrasound fluorescence signal of the first fluorophore. In suchinstances, a method described herein can further comprises (h) combiningthe first modified photoluminescence signal for the first location andthe n additional modified photoluminescent signals for the n additionallocations to generate a spatial plot of ultrasound fluorescence emittedby the first fluorophore within the environment. Additionally, it isalso possible to generate a spatial plot of ultrasound fluorescenceemitted by the second fluorophore within the environment. To generatesuch a plot, the reference signal can be chosen to correspond to thefirst ultrasound fluorescence signal of the second fluorophore, ratherthan of the first fluorophore.

Moreover, in some embodiments described herein, a plurality of differingreference signals can be used to correlate a photoluminescence signaldescribed herein, including in a sequential manner. For example, in someinstances, a method described herein can further comprise (f₂)correlating the first photoluminescence signal with a second referencesignal to generate a second correlation coefficient for the firstlocation, and (g₂) multiplying the first photoluminescence signal by thesecond correlation coefficient for the first location to generate asecond modified photoluminescence signal for the first location. In somecases, the first reference signal corresponds to the first ultrasoundfluorescence signal of the first fluorophore, and the second referencesignal corresponds to the first ultrasound fluorescence signal of thesecond fluorophore.

Not intending to be bound by theory, it is believed that such differingreference signals can be used to obtain multiplexed imaging due to theunique photoluminescence emission profiles of ultrasound-switchablefluorophores, including ultrasound-switchable fluorophores describedherein. Such ultrasound-switchable fluorophores can have uniqueultrasound fluorescence signals. Further, the ultrasound fluorescencesignals can be unique in a wavelength domain and/or in a temporal ortime domain. Thus, in some embodiments described herein, a firstultrasound-switchable fluorophore and a second ultrasound-switchablefluorophore can have differing ultrasound fluorescence emissionprofiles. For example, in some cases, the peak emission wavelengths ofthe profiles differ, such that the fluorophores emit differing colors ofelectromagnetic radiation. In some instances, the fluorophores havediffering temporal intensity decay profiles. More generally, in someembodiments of a method described herein, the ultrasound fluorescenceemission profiles of the first and second fluorophores aremathematically orthogonal or non-correlated or weakly correlated. Insuch instances, when a signal consisting essentially of the ultrasoundfluorescence emission of one fluorophore is correlated with a referencesignal based on the ultrasound fluorescence emission of the otherfluorophore, a small correlation coefficient will be obtained. Forexample, in some cases, correlation of a first ultrasound fluorescencesignal of a first fluorophore using a reference signal corresponding tothe ultrasound fluorescence signal of a second fluorophore will resultin a small correlation coefficient, such as a correlation coefficient ofless than 0.3, and vice versa.

Moreover, it is to be understood that the foregoing steps (e)-(g) ofdetecting and processing a photoluminescence signal at a first locationwithin an environment can be repeated any desired number of times togenerate a plurality of modified photoluminescence signals for aplurality of locations within the environment. For example, in someembodiments, a method described herein using a plurality ofultrasound-switchable fluorophores can further comprise (e_(n))detecting n additional photoluminescence signals at n additionallocations within the environment, wherein the n additionalphotoluminescence signals comprise at least one of an nth additionalultrasound fluorescence signal emitted by the first fluorophore, an nthadditional ultrasound fluorescence signal emitted by the secondfluorophore, and an nth additional background signal. The method canalso comprise (f_(1n)) correlating the n additional photoluminescencesignals with the first reference signal to generate n additional firstcorrelation coefficients for the n additional locations, (g_(1n))multiplying the n additional photoluminescence signals by the nadditional first correlation coefficients for the n additional locationsto generate n additional first modified photoluminescence signals forthe n additional locations, (f_(2n)) correlating the n additionalphotoluminescence signals with the second reference signal to generate nadditional second correlation coefficients for the n additionallocations, and (g_(2n)) multiplying the n additional photoluminescencesignals by the n additional second correlation coefficients for the nadditional locations to generate n additional second modifiedphotoluminescence signals for the n additional locations, wherein n isan integer described above, such as an integer between 1 and 1000.

In addition, as described above for the use of a singleultrasound-switchable fluorophore, a method described herein can be usedto generate a spatial plot of ultrasound fluorescence emission for morethan one fluorophore. For example, in some instances, a method describedherein further comprises (h₁) combining a first modifiedphotoluminescence signal for a first location with n additional firstmodified photoluminescent signals for n additional locations to generatea spatial plot of ultrasound fluorescence emitted by the firstfluorophore within the environment. Moreover, such a method may alsocomprise (h₂) combining a second modified photoluminescence signal forthe first location with n additional second modified photoluminescentsignals for the n additional locations to generate a spatial plot ofultrasound fluorescence emitted by the second fluorophore within theenvironment.

Further, it is to be understood that the same process described abovefor two differing ultrasound-switchable fluorophores can also be usedfor multiplexed imaging using more than two differing fluorophores. Ingeneral, any desired number of differing ultrasound-switchablefluorophores may be used. For example, in some embodiments, three, four,or five differing ultrasound-switchable fluorophores can be used.Moreover, in such cases, a method described herein can comprise carryingout three, four, or five correlating and multiplying steps (such asthose described in steps (f_(1n)), (g_(1n)), (f_(2n)), and (g_(2n)))with three, four, or five reference signals corresponding to the three,four, or five fluorophores, respectively. In general, up to m differingultrasound-switchable fluorophores may be used, wherein m can be 5, 10,20, 50, or 100.

Methods of multiplexed USF imaging described hereinabove may beparticularly useful when no single location within the imagedenvironment contains more than one of the m differingultrasound-switchable fluorophores. However, it is also possible, insome cases, to generate a plurality of spatial plots of ultrasoundfluorescence emitted by a plurality of differing ultrasound-switchablefluorophores, even when the differing fluorophores are present orpossibly present in a common voxel within the environment. For example,in some embodiments, a method described herein comprises (a) disposing afirst ultrasound-switchable fluorophore and a secondultrasound-switchable fluorophore in an environment, (b) exposing theenvironment to an ultrasound beam to create an activation region withinthe environment, (c) disposing the first fluorophore within theactivation region to switch the first fluorophore from an off state toan on state and/or disposing the second fluorophore within theactivation region to switch the second fluorophore from an off state toan on state, and (d) exposing the environment to a beam ofelectromagnetic radiation, thereby exciting the first fluorophore and/orthe second fluorophore. The method can further comprise (e) detecting afirst photoluminescence signal at a first location within theenvironment, wherein the first photoluminescence signal comprises atleast one of a first ultrasound fluorescence signal emitted by the firstfluorophore and a first ultrasound fluorescence signal emitted by thesecond fluorophore. In addition, in some cases, a method describedherein also comprises (f) orthogonally decomposing the firstphotoluminescence signal into a first basis vector corresponding to anormalized ultrasound fluorescence signal of the first fluorophore and asecond basis vector corresponding to a normalized ultrasound signal ofthe second fluorophore. Moreover, in some embodiments, the methodfurther comprises (g₁) determining a basis vector coefficient a for thenormalized ultrasound fluorescence signal of the first fluorophore atthe first location, and (g₂) determining a basis vector coefficient bfor the normalized ultrasound fluorescence signal of the secondfluorophore at the first location. Further, the method can also comprise(h₁) multiplying the normalized ultrasound fluorescence signal of thefirst fluorophore by the coefficient a to generate a separatedultrasound fluorescence signal of the first fluorophore at the firstlocation and (h₂) multiplying the normalized ultrasound fluorescencesignal of the second fluorophore by the coefficient b to generate aseparated ultrasound fluorescence signal of the second fluorophore atthe first location. A “separated” ultrasound fluorescence signal, forreference purposes herein, can refer to an ultrasound fluorescencesignal that has been removed from, dissociated from, or disambiguatedfrom a more complex signal detected by a USF imaging experiment, such asa detected photoluminescence signal described herein that may include acombination of signals, including a combination of differing ultrasoundfluorescence signals. Therefore, carrying out a method in a mannerdescribed herein can permit signals from more than one fluorophorewithin an imaged environment to be distinguished from one another, evenif the fluorophores are present within the same general location withinthe environment, such as within the same voxel.

Moreover, the forgoing process of steps (e)-(h) can be repeated anydesired number of times to generate separated ultrasound fluorescencesignals of the first and/or second fluorophores at any desired number ofadditional locations within the environment. Thus, in some cases, amethod described herein further comprises (e_(n)) detecting n additionalphotoluminescence signals at n additional locations within theenvironment, wherein the n additional photoluminescence signals compriseat least one of an nth additional ultrasound fluorescence signal emittedby the first fluorophore and an nth additional ultrasound fluorescencesignal emitted by the second fluorophore. Such a method can alsocomprise (f_(n)) orthogonally decomposing the n additionalphotoluminescence signals into n additional first basis vectorscorresponding to a normalized ultrasound fluorescence signal of thefirst fluorophore and n additional second basis vectors corresponding toa normalized ultrasound signal of the second fluorophore. In addition,the method can further include (g_(1n)) determining n additional basisvector coefficients a_(n) for the normalized ultrasound fluorescencesignal of the first fluorophore at the n additional locations, (g_(2n))determining n additional basis vector coefficients bn for the normalizedultrasound fluorescence signal of the second fluorophore at the nadditional locations, (h_(1n)) multiplying the normalized ultrasoundfluorescence signal of the first fluorophore by the n additionalcoefficients a_(n) to generate n additional separated ultrasoundfluorescence signals of the first fluorophore at the n additionallocations, and (h_(2n)) multiplying the normalized ultrasoundfluorescence signal of the second fluorophore by the n additionalcoefficients b_(n) to generate n additional separated ultrasoundfluorescence signals of the second fluorophore at the n additionallocations. As described above, n can be any desired integer, such as aninteger between 1 and 1000.

Additionally, as described above, it is also possible to generateseparate spatial plots of ultrasound fluorescence emitted by the firstand second fluorophores within the environment. For example, in someembodiments, a method described herein further comprises (i₁) combiningthe separated ultrasound fluorescence signal of the first fluorophore atthe first location with the n additional separated ultrasoundfluorescence signals of the first fluorophore at the n additionallocations to generate a spatial plot of ultrasound fluorescence emittedby the first fluorophore within the environment and (i₂) combining theseparated ultrasound fluorescence signal of the second fluorophore atthe first location with the n additional separated ultrasoundfluorescence signals of the second fluorophore at the n additionallocations to generate a spatial plot of ultrasound fluorescence emittedby the second fluorophore within the environment.

Moreover, once separated ultrasound fluorescence signals of the firstand second fluorophores are obtained for one or more locations withinthe environment, it is possible, if desired, to improve the SNR of thesesignals in a manner described hereinabove. For example, in someinstances, a method described herein further comprises (j₁) correlatingthe separated ultrasound fluorescence signal of the first fluorophorewith a first reference signal to generate a first correlationcoefficient for the first reference signal for the first location, (k₁)multiplying the separated ultrasound fluorescence signal of the firstfluorophore by the first correlation coefficient for the first referencesignal to generate a first modified separated ultrasound fluorescencesignal of the first fluorophore for the first location, (j₂) correlatingthe separated ultrasound fluorescence signal of the second fluorophorewith a second reference signal to generate a first correlationcoefficient for the second reference signal for the first location, and(k₂) multiplying the separated ultrasound fluorescence signal of thesecond fluorophore by the first correlation coefficient for the secondreference signal to generate a first modified separated ultrasoundfluorescence signal of the second fluorophore for the first location.Further, in such embodiments, the first reference signal can correspondto the first ultrasound fluorescence signal of the first fluorophore,and the second reference signal can correspond to the first ultrasoundfluorescence signal of the second fluorophore.

It is further to be understood that such correlation and modification ofseparated ultrasound fluorescence signals can be carried out for anydesired number of locations within an imaged environment. For example,in some embodiments, a method described herein can comprise (j_(1n))correlating n additional separated ultrasound fluorescence signals ofthe first fluorophore with a first reference signal to generate nadditional correlation coefficients for the first reference signal forthe n additional locations, (k_(1n)) multiplying the n additionalseparated ultrasound fluorescence signals of the first fluorophore bythe n additional correlation coefficients for the first reference signalto generate n additional modified separated ultrasound fluorescencesignals of the first fluorophore for the n additional locations,(j_(2n)) correlating n additional separated ultrasound fluorescencesignals of the second fluorophore with a second reference signal togenerate n additional correlation coefficients for the second referencesignal for the n additional locations, and (k_(2n)) multiplying the nadditional separated ultrasound fluorescence signals of the secondfluorophore by the n additional correlation coefficients for the secondreference signal to generate n additional modified separated ultrasoundfluorescence signals of the second fluorophore for the n additionallocations, wherein the first reference signal corresponds to the firstultrasound fluorescence signal of the first fluorophore, and the secondreference signal corresponds to the first ultrasound fluorescence signalof the second fluorophore.

Moreover, if desired, such modified separated ultrasound fluorescencesignals for a plurality of locations can be used to generate a spatialplot of ultrasound fluorescence emitted by a fluorophore in theenvironment, as described in steps (i₁) or (i₂) above. In addition, itis to be understood that a “modified” ultrasound fluorescence signal canrefer to an ultrasound fluorescence signal that has been modifiedthrough one or more signal processing steps, such as one or moremultiplying steps described herein.

Further, it is to be understood that the same process described abovefor two differing ultrasound-switchable fluorophores can also be usedfor multiplexed imaging using more than two differing fluorophores. Ingeneral, any desired number of differing ultrasound-switchablefluorophores may be used. For example, in some embodiments, three, four,or five differing ultrasound-switchable fluorophores can be used.Moreover, in such cases, a method described herein can comprise carryingout three, four, or five correlating and multiplying steps (such asthose described in steps (f_(1n)), (g_(1n)), (f_(2n)), and (g_(2n)))with three, four, or five reference signals corresponding to the three,four, or five fluorophores, respectively. In general, up to m differingultrasound-switchable fluorophores may be used, wherein m can be 5, 10,20, 50, or 100. However, as described further herein, for suchmultiplexed imaging within a single voxel, it is to be understood thatit is preferred for the m ultrasound fluorescence emission profiles ofthe m differing ultrasound-switchable fluorophores to be mathematicallyorthogonal, non-correlated, or weakly correlated.

Turning now to specific steps of methods, methods of imaging describedherein comprise disposing an ultrasound-switchable fluorophore or apopulation of ultrasound-switchable fluorophores in an environment. Anyenvironment not inconsistent with the objectives of the presentinvention may be used. In some embodiments, the environment is abiological environment. An environment of a method described herein mayalso be a non-biological environment. In some cases, for example, abiological environment is an in vivo environment, such as a tissue,organ, blood vessel, or other portion of a living organism. In someembodiments, the biological environment comprises a tumor or tumorvasculature. In other cases, a biological environment comprises an invitro environment, such as a tissue culture. The biological environmentof a method described herein can also comprise or be replaced by abiological phantom material or tissue-mimicking phantom material, suchas an agar, silicone, polyvinyl alcohol (PVA) gel, polyacrylamide (PAA)gel, or a dispersion of an oil in gelatin. Other phantom materials mayalso be used.

Moreover, in some embodiments, a biological environment comprises deeptissue. “Deep” tissue, for reference purposes herein, comprises tissue(or, in the case of a phantom material, an interior region of thephantom material) that is located at least about 1 cm below the outersurface of the organism, tissue culture, or other larger structureassociated with the biological environment (such as, in the case of aphantom material, the outer surface of the phantom material). In someembodiments, for instance, deep tissue is located between about 1 cm andabout 10 cm or between about 1 cm and about 5 cm below an outer surface.In some cases, deep tissue is located more than 10 cm below an outersurface. Further, an outer surface, in some embodiments, comprises thesurface of the skin of an organism.

In addition, any ultrasound-switchable fluorophore or combination ofdiffering ultrasound-switchable fluorophores not inconsistent with theobjectives of the present invention may be used. An“ultrasound-switchable” fluorophore, for reference purposes herein,comprises a fluorophore operable to switch between an on state and anoff state in response to exposure to an ultrasound beam. The ultrasoundbeam can be either directly or indirectly responsible for the switchingresponse of the fluorophore. For example, in some cases, the ultrasoundbeam interacts directly with the fluorophore, resulting in a switchbetween fluorescence states of the fluorophore. In other cases, theultrasound beam interacts directly with the immediate environment ormicroenvironment of the fluorophore and changes at least one property ofthe fluorophore's microenvironment. In such cases, the fluorophore canswitch between on and off fluorescence states in response to theenvironmental change induced by the ultrasound beam. Thus, thefluorophore can be indirectly switchable in response to exposure to anultrasound beam.

The “on” state of a fluorophore, for reference purposes herein,comprises either (1) a state at which the fluorescence intensity of thefluorophore is relatively high compared to the “off” state of thefluorophore, at which the fluorescence intensity is relatively low; or(2) a state at which the fluorescence lifetime of the fluorophore isrelatively long compared to the “off” state of the fluorophore, at whichthe fluorescence lifetime is relatively short. Further, in both cases,the on and off states substantially define a step function in thefluorescence intensity or lifetime profile when plotted as a function ofa critical switching parameter such as temperature or negative pressure.A fluorophore having a longer lifetime in an on state than an off statecan be particularly suitable for use in methods described herein usingtime-gated or time-delayed detection of emitted photons fromfluorophores, such as time-gated detection in which only those photonsreceived after a relatively long delay following excitation are countedby the detector as part of the USF signal. In some cases, the on stateof a fluorophore exhibits at least about 70 percent, at least about 80percent, or at least about 90 percent of the theoretical maximumfluorescence intensity of the fluorophore, and the off state of thefluorophore exhibits no more than about 50 percent, no more than about30 percent, no more than about 10 percent, or no more than about 5percent of the theoretical maximum fluorescence intensity of thefluorophore.

The physical cause for the existence of an on state versus an off statecan vary. For example, in some cases, the fluorescence intensity orfluorescence lifetime of a fluorophore changes dues to a conformationalor chemical change of the fluorophore in response to a change inenvironmental conditions, such as exhibited by some thermoresponsivepolymers, pH-sensitive chemical species, or pressure sensitivematerials. In some cases, the fluorescence intensity or fluorescencelifetime of a fluorophore changes in response to internal fluorescencequenching, wherein such quenching can be directly or indirectly inducedby the presence of ultrasound.

For example, in some embodiments, a fluorophore described hereincomprises a FRET donor species and a FRET acceptor species, and thedistance between the FRET donor species and the FRET acceptor species isaltered by the presence of an ultrasound beam. The FRET donor speciescan be a first fluorescent species or other chromophore, and the FRETacceptor species can be a second fluorescent species or otherchromophore. In such cases, as understood by one of ordinary skill inthe art, FRET energy transfer between the donor species and the acceptorspecies can result in quenching of the fluorescence of the donorspecies. Thus, the acceptor species can be considered to be afluorescence quenching species of the fluorophore. Any donor-acceptorpair not inconsistent with the objectives of the present invention maybe used in FRET-based fluorophores described herein. For example, insome cases, the donor species comprises Alexa Fluor 546 and the acceptorspecies comprise Alexa Fluor 647. Other combinations of acceptor speciesand donor species are also possible.

In some embodiments, a fluorophore described herein comprises amicrobubble comprising one or more FRET donor species and one or moreFRET acceptor species attached to the exterior surface of themicrobubble, wherein the microbubble is operable to change in size inresponse to the presence of an ultrasound beam. The change in size canincrease or decrease the distance between the FRET donor species and theFRET acceptor species, thus reducing or increasing the FRET energytransfer efficiency. As a result, the fluorescence quenching and theoverall fluorescence intensity of the microbubble can vary based on thesize of the microbubble.

A microbubble described herein can have any size and be formed of anychemical species not inconsistent with the objectives of the presentinvention. In some cases, a microbubble has a diameter between about 1μm and about 10 μm or between about 1 μm and about 5 μm. Other sizes ofmicrobubbles may also be used. Moreover, in some embodiments, amicrobubble described herein comprises a gas core surrounded by a shellformed from a polymeric material, such an organic polymeric material. Inother cases, the shell is formed from a lipid material. In someembodiments, a microbubble comprises a shell formed from one or more ofalbumin, galactose, lipid, and sulfur hexafluoride. In addition, the gascore of a microbubble described herein can comprise one or more of air,nitrogen, and a perfluorocarbon such as octafluoropropane. Moreover, insome cases, a microbubble described herein is formed from a commerciallyavailable microbubble, such as a SonoVue™, Optison™, Imagent™,Definity™, or Targestar™ microbubble. A FRET donor and/or acceptorspecies described herein can be attached to the surface of such amicrobubble in any manner not inconsistent with the objectives of thepresent invention. In some cases, for instance, a donor and/or acceptorspecies is attached to the exterior surface of a commercially availablemicrobubble using one or more of a carbodiimide, maleimide, orbiotin-streptavidin coupling scheme.

In addition, in some embodiments, a fluorophore described hereincomprises a thermoresponsive polymer. A “thermoresponsive” polymer, forreference purposes herein, comprises a polymer having a physical orchemical property that changes in a temperature-dependent manner,wherein the change is a discontinuous or binary change. For example, insome cases, the physical conformation or polarity of a thermoresponsivepolymer changes in a temperature-dependent manner, and thethermoresponsive polymer exhibits a first conformation below a thresholdtemperature and a second, substantially different conformation above thethreshold temperature. In some embodiments, for instance, athermoresponsive polymer exhibits an expanded coil or chain confirmationbelow a threshold temperature and exhibits a compact or globularconformation above the threshold temperature. In some such cases, thethreshold temperature can be referred to as the “lower critical solutiontemperature” (LCST) of the polymer.

Any thermoresponsive polymer not inconsistent with the objectives of thepresent invention may be used. In some embodiments, a thermoresponsivepolymer comprises a poly(N-isopropylacrylamide) or a copolymer ofN-isopropylacrylamide with one or more of acrylamide,N-tert-butylacrylamide, acrylic acid, and allylamine. In other cases, athermoresponsive polymer comprises a poly(N-vinylcaprolacatam) (PVCL) ora poloxamer such as a Pluronic polymer. Other thermoresponsive polymersmay also be used.

Additionally, in some cases, a thermoresponsive polymer of a fluorophoredescribed herein comprises one or more fluorescent moieties or isconjugated to one or more fluorescent species, such as one or morefluorescent dye molecules. The thermoresponsive polymer can beconjugated to the fluorescent species in any manner not inconsistentwith the objectives of the present invention. For example, in somecases, a thermoresponsive polymer is coupled to a fluorescent speciesthrough one or more covalent bonds such as one or more ester bonds orone or more amide bonds.

FIG. 1 schematically illustrates an ultrasound-switched fluorescenceprocess using a thermoresponsive fluorophore according to one embodimentdescribed herein. As illustrated in FIG. 1, a thermoresponsive polymeris conjugated to a fluorescent species to provide a fluorophore. Thefluorophore has a chain conformation and a globular conformationdescribed hereinabove, and the conformation is temperature-dependent.Further, the transition from one conformation to the other results in achange in the fluorescence intensity or lifetime of the fluorescentspecies. As described further herein, the change in fluorescenceintensity or lifetime can be due to differences in the microenvironmentof the fluorescent species when the polymer is in the chain conformationcompared to the globular conformation. For example, in some cases, thepolarity and/or viscosity of the polymer environment experienced by thefluorophore changes depending on whether the polymer is in the chainconformation or the globular conformation.

Further, in some embodiments, a fluorophore described herein comprises afluorescent material dispersed in and/or attached to the surface of athermoresponsive polymer nanoparticle. Moreover, the fluorescenceproperties of the fluorescent material can be dependent on a change ofthe conformation, polarity, or other physical or chemical property ofthe polymer nanoparticle. In addition, the property change can be atemperature-dependent change. In this manner, a change in temperature ofthe thermoresponseive polymer nanoparticle can result in a change influorescence intensity and/or lifetime of the fluorescent material,including a change between an on state of the fluorescent material andan off state of the fluorescent material.

For example, in some embodiments, a thermoresponsive polymernanoparticle can exhibit a temperature-dependent polarity, and thefluorescent material dispersed in the nanoparticle can exhibit apolarity-dependent fluorescence intensity and/or lifetime. Thus, achange in the temperature of the nanoparticle can result in a change inthe fluorescence intensity and/or lifetime of the fluorophore.

In another exemplary embodiment, a thermoresponsive polymer nanoparticlecan have a hydrophilic interior below a threshold temperature and ahydrophobic interior above the threshold temperature. Thus, such ananoparticle can exhibit a temperature-dependent size when dispersed ina polar or non-polar solvent. For example, when dispersed in water oranother polar solvent below the threshold temperature, the nanoparticlecan exhibit a larger size due to the presence of water in thehydrophilic interior of the nanoparticle. Similarly, above the thresholdtemperature, the nanoparticle can exhibit a smaller size due to theexclusion of water from the now hydrophobic interior of thenanoparticle. In this manner, a fluorescent material dispersed in thenanoparticle can have a temperature-dependent concentration, which canresult in temperature-dependent fluorescence properties of the overallfluorophore. This process is illustrated schematically in FIG. 2.

In yet another exemplary embodiment, an ultrasound-switchablefluorophore is formed by incorporating a fluorescent material such as afluorescent dye within the interior of a polymeric nanoparticle ormicelle, such that the polymeric nanoparticle or micelle acts as ananocapsule for the fluorescent material. Moreover, the polymericnanoparticle can be formed from a thermoresponsive polymer, such as athermoresponsive polymer described hereinabove. Non-limiting examples ofpolymers suitable for forming nanocapsules described herein includePluronic F127, Pluronic F98, poly(N-isopropylacrylamide) (PNIPAM), andcopolymers of PNIPAM with acrylamide (AAm) or N-tert-butylacrylamide(TBAm). Moreover, in some instances, a nanoparticle or nanocapsule canbe formed by copolymerizing a thermoresponsive polymer describedhereinabove with a polyethylene glycol (PEG) and/or by conjugating a PEGas a pendant group to a thermoresponsive polymer. Such a fluorophore, insome cases, can have a switching threshold that is controlled at leastin part by the inclusion of PEG, as described further hereinbelow.

A polymer nanoparticle such as a thermoresponsive polymer nanoparticleor a polymer nanocapsule described herein can have any size or shape notinconsistent with the objectives of the present invention. In someembodiments, for instance, a thermoresponsive polymer nanoparticle issubstantially spherical and has a diameter between about 10 nm and about300 nm, between about 50 nm and about 250 nm, between about 50 nm andabout 200 nm, or between about 70 nm and about 150 nm. In some cases, apolymer nanocapsule is substantially spherical and has a diameter ofless than about 100 nm or less than about 50 nm. In some instances, apolymer nanocapsule has a size between about 20 nm and about 90 nm,between about 20 nm and about 80 nm, or between about 20 nm and about 70nm. Other sizes and shapes are also possible.

Further, any fluorescent material not inconsistent with the objectivesof the present invention may be dispersed in and/or attached to athermoresponsive polymer nanoparticle or other polymer nanoparticle toform a fluorophore described herein. In some embodiments, as describedherein, the fluorescent material exhibits a polarity-sensitivefluorescence intensity and/or lifetime. In other cases, the fluorescentmaterial exhibits a temperature-dependent, viscosity-dependent,pH-dependent, and/or an ionic strength-dependent fluorescence intensityand/or lifetime.

Non-limiting examples of fluorescent materials suitable for use in someembodiments described herein include organic dyes such asN,N-dimethyl-4-benzofurazansulfonamide (DBD);4-(2-Aminoethylamino)-7-(N,N-dimethyl sulfamoyl)benzofurazan (DBD-ED);indocyanine green (ICG); a Dylight-700 such as Dylite-700-2B; IR-820;3,3′-Diethylthiatricarbocyanine iodide (DTTCI); LS-277; LS-288; acypate; a rhodamine dye such as rhodamine 6G or rhodamine B; or acoumarin. In some instances, a fluorescent material comprises anazadipyrromethene. In addition, in some cases, a fluorescent materialcomprises an inorganic species such as a semiconductor nanocrystal orquantum dot, including a II-VI semiconductor nanocrystal such as ZnS orCdSe or a III-V semiconductor nanocrystal such as InP or InAs. In otherinstances, a fluorescent material comprises a Lanthanide species.Additional non-limiting examples of fluorescent materials suitable foruse in an ultrasound-switchable fluorophore described herein include thefluorescent materials described in Amin et al., “Syntheses,Electrochemistry, and Photodynamics of Ferrocene-AzadipyrromethaneDonor-Acceptor Dyads and Triads,” J. Phys. Chem. A 2011, 115, 9810-9819;Bandi et al., “A Broad-Band Capturing and Emitting Molecular Triad:Synthesis and Photochemistry,” Chem. Commun., 2013, 49, 2867-2869; Jokicet al., “Highly Photostable Near-Infrared Fluorescent pH Indicators andSensors Based on BF2-Chelated Tetraarylazadipyrromethane Dyes,” Anal.Chem. 2012, 84, 6723-6730; Jiang et al., “A Selective FluorescentTurn-On NIR Probe for Cysteine,” Org. Biomol. Chem., 2012, 10,1966-1968; and Kucukoz et al., “Synthesis, Optical Properties andUltrafast Dynamics of Aza-boron-dipyrromethane Compounds ContainingMethoxy and Hydroxy Groups and Two-Photon Absorption Cross-Section,”Journal of Photochemistry and Photobiology A: Chemistry 247 (2012),24-29; the entireties of which are hereby incorporated by reference.Other fluorescent materials may also be used.

An ultrasound-switchable fluorophore described herein can have anyfluorescence emission profile not inconsistent with the objectives ofthe present invention. For example, in some embodiments, a fluorophoreexhibits an emission profile including visible light or centered in thevisible region of the electromagnetic spectrum, such as between 450 nmand 750 nm. In some cases, a fluorophore exhibits an emission profileincluding infrared (IR) light or centered in the IR region of theelectromagnetic spectrum. For example, in some instances, a fluorophoredescribed herein exhibits an emission profile centered in the near-IR(NIR, 750 nm-1.4 μm), short-wavelength IR (SWIR, 1.4-3 μm),mid-wavelength IR (MWIR, 3-8 μm), or long-wavelength IR (LWIR, 8-15 μm).Moreover, in some embodiments, a fluorophore described herein has anemission profile overlapping with a wavelength at which water and/orbiological tissue has an absorption minimum, such as a wavelengthbetween about 700 nm and about 800 nm or between about 1.25 μm and about1.35 μm. Additionally, in some cases, a population ofultrasound-switchable fluorophores described herein comprisesfluorophores having differing emission profiles. For example, in somecases, a first fluorophore of the population can emit in the NIR and asecond fluorophore of the population can emit in the visible region ofthe electromagnetic spectrum. Moreover, in some instances, a firstfluorophore and a second fluorophore can have differing temporalintensity decay profiles, as described further hereinbelow. In someembodiments, the ultrasound fluorescence emission profiles of a firstfluorophore and a second fluorophore are mathematically orthogonal ornon-correlated. In this manner, multiplexed imaging can be achieved.

Further, in some instances, a fluorophore described herein exhibits afluorescence lifetime of at least about 1 ns, at least about 3 ns, or atleast about 10 ns. In some embodiments, a fluorophore described hereinexhibits a fluorescence lifetime between about 1 ns and about 15 ns,between about 1 ns and about 10 ns, between about 1 ns and about 4 ns,between about 3 ns and about 7 ns, between about 3 ns and about 5 ns, orbetween about 10 ns and about 15 ns.

Additionally, in some embodiments, an ultrasound-switchable fluorophoredescribed herein exhibits one or more desirable features related to theon and off states of the fluorophore. For example, in some cases, afluorophore exhibits a high on-to-off ratio in fluorescence intensity(I_(On)/I_(Off)), a high on-to-off ratio in fluorescence lifetime(τ_(On)/τ_(Off)), a sharp transition bandwidth between on and off states(T_(BW)), and/or an adjustable switching threshold (S_(th)), such as anadjustable switching threshold temperature (T_(th)) or an adjustableswitching threshold pressure (P_(th)). These metrics can be furtherdescribed with reference to FIG. 3.

FIG. 3 illustrates plots of the fluorescence intensity and fluorescencelifetime of a temperature-dependent fluorophore as a function oftemperature. However, it is to be understood that the same principlesand nomenclature can be applied in an analogous way for a fluorophorethat exhibits pressure-dependent fluorescence or fluorescence dependenton some other variable described herein. In such an instance, thetemperature axis of FIG. 3 could be replaced by a pressure axis or anaxis corresponding to another variable related to fluorescence switchingwithout otherwise substantially altering the appearance of FIG. 3. Withreference to FIG. 3, T_(th) refers to the switching thresholdtemperature. I_(On)/I_(Off) refers to the ratio of the averagefluorescence intensity of the fluorophore over a range of temperaturesabove the threshold temperature to the average fluorescence intensity ofthe fluorophore over a range of temperatures below the thresholdtemperature. Similarly, τ_(On)/τ_(Off) refers to the ratio of theaverage fluorescence lifetime of the fluorophore over a range oftemperatures above the threshold temperature to the average fluorescencelifetime of the fluorophore over a range of temperatures below thethreshold temperature. In some embodiments, the averages are taken overa range of temperatures having a magnitude that is about 5 percent toabout 100 percent of the magnitude of the switching threshold value butthat lie outside of the transition bandwith T_(BW). T_(BW) refers to therange of temperature values (or, analogously, pressure or other variablevalues) over which the fluorphore switches from the on state to the offstate in the manner of a step function. In other words, T_(BW) refers tothe width of the step between the on and off states. The smaller theT_(BW), the more the fluorescence intensity profile of the fluorophoreresembles a true step function having a discontinuity between the onstate and the off state. In FIG. 3, the I_(on) value is taken as theaverage intensity over a temperature range of about 33° C. to about 48°C. (a range of about 16° C., or about 62 percent of the T_(th) value of26° C.) and the I_(Off) value is taken as the average intensity over atemperature range of about 23° C. to about 25° C. (a range of about 3°C., or about 12 percent of the T_(th) value of 26° C.). In general, therange of temperature values used for determining the averagefluorescence intensity in the on and off states can be based on therange of temperature values of interest for a particular imagingapplication. An ultrasound-switchable fluorophore described herein canexhibit any of the I_(on)/I_(Off), τ_(On)/τ_(Off), T_(BW), and T_(th)values provided hereinbelow in Table 1.

TABLE 1 I_(On)/I_(Off) τ_(On)/τ_(Off) T_(BW) (° C.) T_(th) (° C.) >2 ≥2<15 >25 >3 ≥3 <10 >30 >5 ≥5 ≤5 >37 >8  2-10 1-15 ≥40 2-10 2-7 1-10 20-453-10 2-5 3-12 25-35 3-9  3-7 3-10 37-45 5-9  3-5 3-5  38-45

Methods of imaging described herein, in some embodiments, also compriseexposing an environment such as a biological environment to a pulsedbeam of electromagnetic radiation, including prior to exposing thebiological environment to an ultrasound beam. The pulsed beam ofelectromagnetic radiation can have a picosecond pulse duration, such asa pulse duration of no greater than 100 ps, wherein the pulse durationis defined as the FWHM of the optical power of the pulsed beam overtime. The pulsed beam can have any wavelength and power not inconsistentwith the objectives of the present invention. In some cases, forinstance, the wavelength of the pulsed beam is selected to substantiallyoverlap with the absorption profile of one or more species present inthe biological environment, as further described hereinabove. In someembodiments, the pulsed beam has a visible wavelength or a NIRwavelength. Other pulsed beams may also be used.

Methods of imaging described herein also comprise exposing anenvironment such as a biological environment to one or more ultrasoundbeams to create an activation region within the environment. Theultrasound beam can have any ultrasound frequency not inconsistent withthe objectives of the present invention. In some embodiments, anultrasound beam comprises an oscillating sound pressure wave with afrequency of greater than about 20 kHz or greater than about 2 MHz. Insome cases, an ultrasound beam described herein has a frequency of up toabout 5 GHz or up to about 3 GHz. In some embodiments, an ultrasoundbeam has a frequency between about 20 kHz and about 5 GHz, between about50 kHz and about 1 GHz, between about 500 kHz and about 4 GHz, betweenabout 1 MHz and about 5 GHz, between about 2 MHz and about 20 MHz,between about 2 MHz and about 10 MHz, between about 5 MHz and about 200MHz, between about 5 MHz and about 15 MHz, between about 200 MHz andabout 1 GHz, between about 500 MHz and about 5 GHz, or between about 1GHz and about 5 GHz.

In addition, an ultrasound beam can have any power not inconsistent withthe objectives of the present invention. In some embodiments, forinstance, an ultrasound beam has a power between about 0.1 W/cm² andabout 10 W/cm², between about 0.1 W/cm² and about 5 W/cm², between about0.5 W/cm² and about 5 W/cm², between about 1 W/cm² and about 10 W/cm²,or between about 1 W/cm² and about 5 W/cm². In other cases, anultrasound beam has a power between about 100 W/cm² and about 5000W/cm², or between about 100 W/cm² and about 3000 W/cm². In some cases,the use of an ultrasound beam having a high power, such as a powerdescribed herein, can result in the generation of non-linear effectswithin the activation region. Moreover, in some embodiments, theeffective size of the activation region can be reduced in this manner,leading to improved imaging resolution.

An environment can be exposed to an ultrasound beam in any manner notinconsistent with the objectives of the present invention. For example,in some embodiments, a biological environment is exposed to anultrasound beam described herein for only a limited duration. In somecases, for instance, the ultrasound beam is provided to the environmentfor less than about 1 second or less than about 500 ms. In someembodiments, the ultrasound beam is provided to the environment for lessthan about 300 ms, less than about 100 ms, less than about 50 ms, orless than about 10 ms. In some cases, the ultrasound beam is provided tothe environment for about 1 ms to about 1 second, about 1 ms to about500 ms, about 1 ms to about 300 ms, about 1 ms to about 100 ms, about 1ms to about 50 ms, about 1 ms to about 10 ms, about 10 ms to about 300ms, about 10 ms to about 100 ms, about 10 ms to about 50 ms, or about 50ms to about 100 ms. The use of short exposure times of a biologicalenvironment to an ultrasound beam, in some embodiments, can permit thetime-gating of fluorescence signals, such that a desired USF signal canbe temporally separated from one or more undesired or non-analytefluorescence signals, such as a tissue autofluorescence signal or asignal from a randomly switched-on fluorophore.

Moreover, the ultrasound beam can be a continuous wave beam or a pulsedor modulated beam. The use of a modulated or pulsed ultrasound beam, insome embodiments, can further improve the SNR of a method describedherein by permitting frequency-gated detection of the USF signal. Forexample, in some cases, a pulsed or modulated ultrasound beam providesan ultrasound exposure having a specific frequency or modulation. As aresult, the corresponding USF signal can also exhibit the same specificfrequency or modulation. Thus, in some such cases, a lock-in amplifieris used to increase the sensitivity of the detector to the specificfrequency or modulation, thus increasing the overall sensitivity and SNRof the method.

In some embodiments of methods described herein, a single ultrasoundbeam is directed toward the environment using a single ultrasoundtransducer, such as a high intensity focused ultrasound (HIFU)transducer. In other instances, a plurality of ultrasound beams isdirected toward the environment using a plurality of ultrasoundtransducers. Moreover, in some cases, a first ultrasound beam isdirected toward the environment at a first angle and/or from a firstdirection, and a second ultrasound beam is directed toward theenvironment at a second angle and/or from a second direction differingfrom the first angle and/or direction. In some embodiments, forinstance, the first and second directions are orthogonal orsubstantially orthogonal directions, such as directions separated by 80to 100 degrees. In other cases, the directions are separated by lessthan 80 degrees or more than 100 degrees. Further, if desired,additional ultrasound beams may also be directed toward the environmentfrom additional directions or at additional angles. In such cases, thefocal zones of the beams can overlap or intersect with one another toform an activation region at the intersection of the beams. In thismanner, an activation region can have a smaller volume or cross sectionthan the focal zone or cross section of a single ultrasound beam used togenerate the activation region, thereby improving imaging resolution. Insome cases, for instance, the activation region has a lateral dimensionand/or an axial dimension of less than about 2 mm, less than 1.5 mm, orless than about 1 mm. In some embodiments, the activation region has alateral dimension and/or an axial dimension of less than about 700 μm orless than about 500 μm. In some embodiments, the activation region has alateral dimension and/or an axial dimension of about 300 μm to about 2mm, about 400 μm to about 1.5 mm, about 400 μm to about 1 mm, about 400μm to about 700 μm, or about 400 μm to about 500 μm. In some cases, thelateral and axial dimensions both have a size recited herein, includinga size below about 1 mm or below about 700 μm. Moreover, in someembodiments, the lateral and axial dimensions of the activation regionare different, thereby providing a relatively anisotropic activationregion. Alternatively, in other instances, the lateral and axialdimensions are substantially the same, thereby providing a relatively“square” or isotropic activation region.

An “activation region,” for reference purposes herein, comprises aregion of the environment in which ultrasound-switchable fluorophoresdescribed herein can be switched from an off state to an on state. Forexample, in some cases, an activation region comprises a region ofnegative pressure compared to other portions of the environment.Similarly, in other instances, an activation region comprises a hightemperature region. As described further herein, the temperature,pressure, or other characteristic of an activation region describedherein can be selected based on the switching threshold of a fluorophoredisposed in the biological environment. For example, in some cases, oneor more ultrasound beams are configured to form an activation regionhaving an average temperature or a maximum temperature greater thanabout 30° C., greater than about 35° C., or between about 30° C. andabout 50° C. In other embodiments, an activation region has an averagenegative pressure or a maximum negative pressure between about 10 kPaand about 150 kPa or between about 80 kPa and about 120 kPa. Moreover,as described further herein, the size, shape, and/or other properties ofthe activation region can be determined by the number and/or power ofthe one or more ultrasound beams used to form the activation region. Insome cases, for instance, the size and shape of an activation region isdefined by the focal zone of a single ultrasound beam. In other cases,an activation region is defined by the overlap of the focal zones of aplurality of ultrasound beams.

A fluorophore described herein can be disposed within an activationregion in any manner not inconsistent with the objectives of the presentinvention. In some cases, a fluorophore enters or is disposed within anactivation region of an environment by diffusing into the activationregion from an adjacent area of the environment. In other instances, anactivation region is created within a specific location within anenvironment where it is known that a fluorophore or population offluorophores is likely to be found or may be found. For example, in someembodiments, an ultrasound beam described herein is raster scannedacross or within an environment, thereby producing a plurality ofactivation regions in different locations within the environment in asequential manner.

Methods of imaging described herein also comprise exposing anenvironment to a beam of electromagnetic radiation and/or exciting atleast one fluorophore in an on state with a beam of electromagneticradiation. A fluorophore can be excited with a beam of electromagneticradiation in any manner not inconsistent with the objectives of thepresent invention. In some embodiments, for instance, a fluorophore isexcited using a laser excitation source such as a diode laser. In otherinstances, a fluorophore is excited using one or more light emittingdiodes (LEDs) or a broadband excitation source. Moreover, an excitationsource described herein can provide any wavelength of light notinconsistent with the objectives of the present invention. In someembodiments, a fluorophore described herein is excited with a beam ofelectromagnetic radiation comprising visible light, MR light, or IRlight. In other cases, the beam of electromagnetic radiation comprisesultraviolet (UV) light.

Methods described herein also comprise detecting a photoluminescencesignal or other light emitted within an environment or within a specificlocation within an environment. In some embodiments, for instance, amethod comprises detecting light emitted by at least oneultrasound-switchable fluorophore. Light emitted by the fluorophore canbe detected in any manner not inconsistent with the objectives of thepresent invention. In some embodiments, for example, detecting lightemitted by at least one fluorophore in an on state comprises detectingthe light in a time-gated or frequency-gated manner, including atime-gated manner or frequency-gated manner described herein. In somecases, the light emitted by the at least one fluorophore in the on stateis detected after a time delay that is longer than the fluorescencelifetime of the fluorophore in the off state or longer than thefluorescence lifetime of another species present in the biologicalenvironment. For example, in some embodiments, the light emitted by theat least one fluorophore in the on state is detected after a time delaythat is longer than the autofluorescence lifetime of a non-fluorophorespecies present in the biological environment, such as theautofluorescence lifetime of tissue, which may be up to about 4 ns or upto about 5 ns. In addition, any detector not inconsistent with theobjectives of the present invention may be used. In some embodiments,for instance, one or more photomultiplier tube (PMT) detectors can beused. Other configurations are also possible.

Similarly, detecting a photoluminescence signal at one or more locationswithin an environment can be carried out in any manner not inconsistentwith the objectives of the present invention. In some cases, forexample, a plurality of photoluminescence signals at a plurality oflocations within an environment is detected by raster scanning theenvironment. Such raster scanning can include raster scanning of one ormore ultrasound beams across or within the environment, such that theultrasound beam sequentially generates a series of activation regions atdifferent locations within the environment. It is also possible, in someinstances, to move or scan a detector described herein from location tolocation within the environment. Moving or scanning a detector in such amanner can increase the detection area of the method. In other cases, atwo-dimensional detector such as a charge-coupled device (CCD) imagesensor or camera is used to detect photoluminescence signals at aplurality of locations simultaneously.

Methods of imaging described herein, in some embodiments, also comprisecorrelating one or more detected photoluminescence signals with one ormore reference signals to generate one or more correlation coefficientsfor one or more locations within an imaged environment. Such methods canalso comprise multiplying the one or more detected photoluminescencesignals by the one or more correlation coefficients for the one or morelocations to generate one or more modified photoluminescence signals forthe one or more locations. The foregoing correlation and multiplicationsteps can be carried out in any manner not inconsistent with theobjectives of the present invention. Moreover, a “reference” signal of amethod described herein can be a signal that has the same luminescenceor fluorescence emission profile as a fluorophore disposed in the imagedenvironment. Thus, a “reference” signal can be used as a standard signalagainst which a detected signal is compared, as described furtherherein. In addition, it is to be understood that such a reference signalcan be generated or measured under the same or substantially similarexperimental conditions as used when the one or more photoluminescencesignals are detected from the imaged environment. For instance, thefollowing experimental conditions can be held constant or substantiallyconstant (within experimental error) for the generation or measurementof a reference signal and the detection of one or more photoluminescencesignals: the manner of exposing the environment to an ultrasound beam(e.g., the number, power, and orientation of ultrasound beams used); themanner of exposing the environment to a beam of electromagneticradiation (e.g., the power, wavelength, and type (pulsed or continuouswave) of radiation source used); the manner of detectingphotoluminescence signals (e.g., the type and placement of the detectorused); and the nature of the environment (e.g., the depth of imaging andtype of tissue used).

In some embodiments described herein, correlating a photoluminescencesignal with a reference signal comprises comparing a temporal intensitydecay profile of the photoluminescence signal to a temporal intensitydecay profile of the reference signal. Such a comparison, in someinstances, can generate a correlation coefficient that serves as ametric of how closely a detected photoluminescence signal corresponds tothe reference signal. Not intending to be bound by theory, it isbelieved that the unique spectral signatures of ultrasound-switchablefluorophores in the time domain can permit the generation of especiallyuseful correlation coefficients when the temporal intensity decayprofiles of the photoluminescence signal and reference signal arecompared, as described further hereinbelow. In some embodiments, acorrelation coefficient for a location within an environment isgenerated according to Equation (1):

$\begin{matrix}{{\rho_{I,R} = \frac{( {\sum{{I(t)}{R(t)}}} ) - \frac{( {\sum{I(t)}} )( {\sum{R(t)}} )}{N}}{\sqrt{( {{\sum{I(t)}^{2}} - \frac{( {\sum{I(t)}} )^{2}}{N}} )( {{\sum{R(t)}^{2}} - \frac{( {\sum{R(t)}} )^{2}}{N}} )}}},} & (1)\end{matrix}$

wherein ρ_(I,R) is the correlation coefficient for the first location,I(t) is the temporal intensity decay profile of the firstphotoluminescence signal at the first location, R(t) is the temporalintensity decay profile of the first reference signal, and N is thenumber of the time point in I(t) and R(t).

Moreover, in some cases, a correlation coefficient generated in a mannerdescribed herein is a binned correlation coefficient. A “binned”correlation coefficient, for reference purposes herein, is a correlationcoefficient that is assigned a value based on a binning of all possiblecorrelation coefficient values. For example, in some cases, the value ofa correlation coefficient can first be determined according to Equation(1) hereinabove. By using Equation (1), all correlation coefficients cantheoretically have a value between −1 and +1. However, for a correlationstep described herein, negative values may not be physically meaningful.Therefore, if Equation (1) provides a negative value of a correlationcoefficient, the binning process can be used to force the negativecorrelation coefficient to have a value of zero. Other values ofcorrelation coefficients may also be “altered” or binned to provide animproved SNR. Thus, in some cases, as a next step in the binningprocess, the “actual” value of a specific correlation coefficient can beused to place the specific correlation coefficient in one of a pluralityof “bins” of coefficient values. In addition, for purposes of carryingout a multiplication step described herein, such as step (g) or (g_(n))above, all correlation coefficients in the same bin can be treated ashaving the same value, such as a value provided by a binning table usedto bin the correlation coefficients. One non-limiting example of abinning table suitable for use in some embodiments described herein isprovided below in Table 2. However, it is to be understood that otherbinning tables may also be used.

TABLE 2 Value (x) of Value to be Used for Correlation CoefficientStrength of Correlation Multiplication Steps x < 0 Anti-Correlation 0 x= 0 Zero Correlation 0   0 < x < 0.3 Weak Correlation 0 0.3 ≤ x < 0.9Moderate Correlation  x³ 0.9 < x   Strong Correlation x

In addition, it is further to be understood that a correlating and/ormultiplying step described herein can be carried out using any computeror software algorithm or other hardware and/or software not inconsistentwith the objectives of the present invention. In some cases, forinstance, one or more MATLAB algorithms are used.

Methods described herein, in some embodiments, also comprise combining aplurality of modified photoluminescence signals for a plurality oflocations within an environment to generate a spatial plot of ultrasoundfluorescence emitted by a fluorophore within the environment. Themodified photoluminescence signals can be combined in any manner notinconsistent with the objectives of the present invention. In somecases, for example, the modified photoluminescence signals are combinedto provide a plot of fluorescence intensity as a function of distancealong one or more axes. Plots of fluorescence intensity as a function ofdistance in one dimension are illustrated, for instance, in FIG. 22. Inother embodiments, modified photoluminescence signals are combined toprovide a plot of fluorescence intensity as a function of two dimensionsor three dimensions within the environment. Modified photoluminescencesignals can be combined in other manners as well.

Further, as described above, a spatial plot generated according to amethod described herein can have an improved SNR, including compared toan otherwise similar plot generated without carrying out the correlatingand multiplying steps described herein. For example, in some cases, aspatial plot of ultrasound fluorescence emitted by a fluorophore withinan environment described herein can have an SNR that is at least about50% greater, at least about 70% greater, at least about 100% greater, atleast about 150% greater, at least about 200% greater, at least about250% greater, at least about 300% greater, at least about 400% greater,at least about 500% greater, at least about 600% greater, at least about700% greater, at least about 800% greater, at least about 900% greater,or at least about 1000% greater than the SNR of a spatial plot ofultrasound fluorescence emitted by the fluorophore within theenvironment (or a similar environment) when a correlating step is notcarried out in a manner described herein. It is to be understood thatthe foregoing percentages are determined by dividing the difference inthe two relevant SNR values by the lower SNR value, and then multiplyingby 100. In some instances, the SNR obtained using a method describedherein is about 20-300% greater, about 15-250% greater, about 15-200%greater, about 15-100% greater, about 50-300% greater, about 50-250%greater, about 50-200% greater, about 50-150% greater, about 50-100%greater, about 70-300% greater, about 70-250% greater, about 100-300%greater, about 100-250% greater, about 100-200% greater, about 150-300%greater, about 150-250% greater, about 200-300% greater, or about200-250% greater than the SNR obtained by an otherwise similar methodthat does not include a correlating step described herein. Moreover, insome embodiments, a spatial plot of ultrasound fluorescence emitted by afluorophore within an environment described herein can have an SNR of atleast about 80, at least about 100, at least about 150, at least about200, at least about 250, at least about 300, at least about 350, or atleast about 400. In some instances, the SNR of a spatial plot generatedin a manner described herein is between about 80 and about 400, betweenabout 100 and about 350, between about 150 and about 350, between about200 and about 350, between about 250 and about 350, between about 300and about 400, or between about 300 and about 350. Non-limiting examplesof spatial plots provided in a manner described herein are illustratedand further described in Example 10 hereinbelow.

It is further to be noted that the value of the SNR of a spatial plotdescribed herein can be determined by in any manner not inconsistentwith the objectives of the present invention. In some cases, the SNR ofa spatial plot is determined as follows. First, using the emissionprofile of a normalized USF image, the peak intensity and the FWHM ofthe profile are determined. The peak wavelength is then assigned as thecenter of a signal range having a bandwidth that is three times thevalue of the FWHM. Next, all of the data outside this signal range istreated as background. The standard deviation of the background is thencalculated and treated as the noise of the emission profile. The SNR ofthe profile can then be calculated by dividing the peak intensity by thenoise. Further, if desired, the SNR for a plurality of measurements canbe determined and averaged. The average SNR can then be taken as the SNRof the USF image.

In addition, methods described herein, in some cases, compriseorthogonally decomposing one or more photoluminescence signals into afirst basis vector corresponding to a normalized ultrasound fluorescencesignal of a first fluorophore and a second basis vector corresponding toa normalized ultrasound signal of a second fluorophore. Such methods canalso comprise determining basis vector coefficients for normalizedultrasound fluorescence signals of a plurality of fluorophores at aplurality of locations within an environment. A photoluminescence signalcan be decomposed in any manner not inconsistent with the objectives ofthe present invention. For example, in some instances, aphotoluminescence signal is decomposed into basis vectors using acomputer or software algorithm or other hardware such as a MATLAB curvefitting algorithm. Similarly, once the photoluminescence signal isdecomposed into basis vectors, the coefficients of the basis vectors canbe determined using any suitable computer or software algorithm, such asa MATLAB algorithm, as described further hereinbelow. In addition,multiplying a normalized ultrasound fluorescence signal of a fluorophoreby the appropriate coefficient to generate a separated ultrasoundfluorescence signal of the fluorophore at a specific location within theenvironment can be carried out in any manner not inconsistent with theobjectives of the present invention. For example, in some instances, analgorithm such as a MATLAB algorithm may be used.

Methods described herein, in some cases, also comprise combiningseparated ultrasound fluorescence signals of one or more fluorophores ata plurality of locations to generate a spatial plot of ultrasoundfluorescence emitted by the one or more fluorophores within theenvironment. The separated ultrasound fluorescence signals can becombined in any manner not inconsistent with the objectives of thepresent invention. In some cases, for example, the separated signals arecombined to provide a plot of fluorescence intensity as a function ofdistance along one or more axes. In other embodiments, separated signalsare combined to provide a plot of fluorescence intensity as a functionof two dimensions or three dimensions within the environment. Combiningseparated ultrasound fluorescence signals in a manner described hereincan thus provide multiplexed imaging of a plurality of fluorophores inone, two, or three dimensions within an environment.

As described hereinabove, methods of imaging described herein, in someembodiments, can exhibit improved penetration depth/resolution ratios(DRRs). The “penetration depth” of an imaging method, for referencepurposes herein, is defined as the depth below the surface of an imagedobject at which the intensity of the ultrasound beam inside the objectfalls to 1/e (about 37 percent) of its initial value at the surface. The“resolution” of a method, for reference purposes herein, is themicroscopic resolution (i.e., the size at which separate objects can bedistinguished), which is taken to be equal to the FWHM of the activationregion in a given dimension. In some embodiments, a method describedherein exhibits a DRR of at least about 100. In other cases, a methoddescribed herein exhibits a DRR of at least about 200, at least about300, or at least about 400. In some embodiments, a method describedherein exhibits a DRR of up to about 500. In some cases, a methoddescribed herein exhibits a DRR between about 100 and about 500, betweenabout 100 and about 400, between about 100 and about 300, or betweenabout 200 and about 500. Further, the penetration depth of a methoddescribed herein, in some embodiments, can be up to 100 mm, up to 50 mm,or up to 30 mm. In some cases, the penetration depth is between about 10mm and about 100 mm, between about 10 mm and about 60 mm, between about10 mm and about 50 mm, between about 20 mm and about 90 mm, or betweenabout 20 mm and about 50 mm. In addition, the resolution of a methoddescribed herein, in some embodiments, is less than about 100 μm, lessthan about 70 μm, less than about 50 μm, or less than about 30 μm. Insome cases, the resolution is between about 10 μm and about 100 μm,between about 10 μm and about 70 μm, between about 10 μm and about 50μm, between about 10 μm and about 30 μm, between about 20 μm and about100 μm, between about 20 μm and about 80 μm, between about 20 μm andabout 50 μm, or between about 30 μm and about 70 μm.

It is to be understood that a method of imaging described herein caninclude any combination of steps described herein and use anycombination of equipment and materials described herein not inconsistentwith the objectives of the present invention. For example, in somecases, a method described herein comprises disposing one or morefluorophores comprising a thermoresponsive polymer in deep biologicaltissue, forming an activation region using two orthogonal HIFUtransducers, and detecting emission from the fluorophores in atime-gated manner, thereby providing a DRR greater than about 200.Moreover, in some instances, such a method further comprises generatinga spatial plot of the ultrasound fluorescence emission of thefluorophores after generating a plurality of correlation coefficientsfor a plurality of locations within the environment, and/or afterorthogonally decomposing one or more photoluminescence signals andsubsequently generating separated ultrasound fluorescence signals for aplurality of fluorophores. Other combinations and configurations arealso possible.

Some embodiments described herein are further illustrated in thefollowing non-limiting examples.

Example 1 Ultrasound-Switchable Fluorophores General

A series of ultrasound-switchable fluorophores or contrast agentssuitable for use in methods of imaging according to some embodimentsdescribed herein was prepared by encapsulating an environment-sensitiveNIR dye, indocyanine green (ICG), into thermoresponsive polymernanoparticles (NPs). The NPs can be disposed in an aqueous environmentsuch as a biological environment described herein. When theenvironment's temperature is below a threshold temperature (which can bereferred to as LCST of the NPs), the NPs are hydrophilic and absorb alarge amount of water, with the result that the NPs have a relativelylarge average diameter. Not intending to be bound by theory, it isbelieved that ICG molecules fluoresce weakly in water-richmicroenvironments because water provides a polar and nonviscousmicroenvironment and thereby increases the nonradiative decay rate ofthe excited ICG molecules. When the temperature increases above thethreshold temperature, the NPs become hydrophobic, causing expulsion ofwater from the NPs and a corresponding reduction in average NP diameter.Again not intending to be bound by theory, it is believed that the ICGmolecules dispersed within the NPs are thus exposed to a polymer-richmicroenvironment having a relatively low polarity and high viscositycompared to the water-rich microenvironment. It is believed that such alow polarity, high viscosity microenvironment can suppress thenonradiative decay rate of the excited ICG molecules, resulting in anincrease in the fluorescence intensity of the ICG. It was observed thatthis fluorescence switching behavior from an off state to an on statewas reversible and repeatable. In particular, a high intensity focusedultrasound (HIFU) transducer could be used to reversibly and repeatedlyswitch the fluorophores between on and off states by altering thetemperature in the transducer's focal zone above and below the LCST ofthe NPs.

The NPs were formed from thermoresponsive polymers of either poly(N-isopropylacrylamide) (PNIPAM) or its copolymer with acrylamide (AAm)or N-tert-butylacrylamide (TBAm). FIG. 4 illustrates the structures ofsuch polymers and ICG. Specifically, four types of polymer NPs weresynthesized, including (1) ICG-encapsulated P(NIPAM-TBAm185:15) NPs, (2)ICG-encapsulated PNIPAM NPs, (3) ICG-encapsulated P(NIPAM-AAm 90:10)NPs, and (4) ICG-encapsulated P(NIPAM-AAm 86:14) NPs. The ratios in theforegoing formulas refer to the molar ratio between the monomer of NIPAMand the monomer of TBAm or AAm used to form the NPs. The LCST of thesethermoresponsive polymer NPs could be altered based on the amount of AAmand/or TBAm copolymerized with PNIPAM. For example, using a hydrophilicmonomer (such as AAm) resulted in a polymer having a higher LCST. Incontrast, using a hydrophobic monomer (such as TBAm) decreased the LCST.In addition, it should be noted that TBAm is more hydrophobic thanNIPAM, while AAm is more hydrophilic than NIPAM.

Materials

N-isopropylacrylamide (NIPAM), acrylamide (AAm), ammonium persulfate(APS), sodium dodecyl sulfate (SDS), N,N,N′,N′-tetramethyl ethylenediamine (TEMED), N,N′-methylenebisacrylamide (BIS),N-tert-butylacrylamide (TBAm), sodium ascorbate, and ICG were purchasedfrom Sigma-Aldrich (St. Louis, Mo., USA). All chemicals were used asreceived without further purification.

Synthesis

The ICG-containing PNIPAM NPs were prepared as follows. Other NPs wereprepared using a similar protocol, except with the addition ofappropriate amounts of TBAm and/or AAm monomers. In addition, it wasalso possible to partially or completely replace TBAm and/or AAm withone or more of acrylic acid (AAc) and allylamine (AH) to form othercopolymers of NIPAM, such as P(NIPAM-AAc), P(TBAm-NIPAM-AAc), orP(NIPAM-AH).

Briefly, to prepare PNIPAM NPs, NIPAM (monomer, 0.6822 g), BIS (crosslinker, 0.0131 g), and SDS (surfactant, 0.0219 g) were dissolved in 50mL deionized water in a 250 mL Schlenk tube, followed by purging withnitrogen for 10 minutes. ICG (fluorophore, 0.0034 g), APS (initiator,0.039 g), and TEMED (accelerator, 51 μL) were then added into the tube.The tube was then placed under an inert nitrogen atmosphere by threecycles of applying vacuum on a Schlenk line followed by backfilling withnitrogen. The contents of the flask were then stirred at roomtemperature for 4 hours. The reaction was stopped by exposing the flaskcontents to air. The product was dialyzed against deionized water usinga 10-kDa molecular weight cutoff membrane for 3 days to remove extrasurfactants and unreacted materials. The composition of the finalproduct was confirmed with a Fourier transform infrared (FTIR)spectrometer (Thermo Nicolet 6700, West Palm Beach, Fla., USA), at 4,000to 600 cm⁻¹.

The diameter of the NPs was measured by dynamic light scattering (DLS)and transmission electron microscopy (TEM). TEM was also used todetermine the morphology of the NPs. For DLS measurements, 200 μL of theproduct was diluted with 2.8 mL of deionized water and then analyzed atroom temperature (25° C.) with a Nanotrac 150 (Microtrac, Inc., Nikkiso,San Diego, Calif., USA). For TEM measurements, samples were prepared bydrop casting an aqueous dispersion of product NPs (at about 1 mg/mL)onto a carbon-coated copper grid (FF200-Cu-50, Electron MicroscopySciences, Hatfield, Pa., USA), followed by staining with 0.2% uranylacetate. TEM experiments were carried out using a JEOL 1200 EX TEM(JEOL, Peabody, Mass., USA). The NPs had sizes between 70 nm and 150 nm,based on dynamic light scattering (DLS) and transmission electronmicroscopy (TEM). In particular, when measured by DLS, (1)ICG-containing PNIPAM NPs had an average size of 150±25 nm; (2)ICG-containing P(NIPAM-TBAm 185:15) NPs had an average size of 76±4 nm;(3) ICG-containing P(NIPAM-AAm 86:14) NPs had an average size of 75±25nm; and (4) ICG-containing P(NIPAM-AAm 90:10) NPs had an average size ofsize of 76±2 nm. The sizes measured by DLS were somewhat larger than thesizes measured by TEM due to the presence of surfactant (SDS) andhydration layers around the NPs in aqueous solution. For example, theaverage size of the NPs of sample (1) above was approximately 110 nmwhen measured by TEM. The NPs were nearly spherical.

The fluorescence switching curves of the polymer NPs are shown in FIG.5. The fluorescence intensity is plotted as a function of the sampletemperature. The sharp switching features can be clearly seen for allfour NPs, with switching threshold temperatures (LCSTs or T_(th)'s) of28° C., 31° C., 37° C., and 41° C. Further, switching was observedmultiple times in a single sample. For example, FIG. 6 illustratesfluorescence data for ICG-containing P(NIPAM-AAm 90:10) NPs at 12different time points (measurement points 1-12 on the x-axis) cyclingbetween low temperature (25° C., measurement points 1, 3, 5, 7, 9, and11) and high temperature (44° C., measurement points 2, 4, 6, 8, 10, and12). In addition, the I_(On)/I_(Off) ratio reached 3.3, 2.9, 9.1, and9.1, respectively, for samples (1), (2), (3), and (4). These values areat least 1.6 to 5.1 times higher than that of some other contrastagents.

The system used to measure the fluorescence characteristics offluorophores described herein is illustrated schematically in FIG. 7. Ingeneral, the emission pulses of fluorophores were averaged 100 times andthe averaged peak value was used to represent the fluorescenceintensity. As illustrated in FIG. 7, “F_(ex)” refers to an excitationfilter; “F_(em)” refers to an emission filter; “L” refers to a lens;“PMT” refers to a photomultiplier tube; “BS” refers to a beam splitter;“PD” refers to a photodiode; “PDG” refers to a pulse-delay generator;and “ND filter” refers to a natural density filter.

Example 2 Methods of Imaging Using Thermoresponsive PolymerNanoparticles General

Methods of imaging according to some embodiments described herein werecarried out as follows. First, a small silicone tube (with a meandiameter of 0.69 mm; Instech Lab, BSILT031, PA, USA) was filled with anaqueous solution of the ICG-containing PNIPAM NPs (LCST=31° C.) ofExample 1. The tube was then embedded in a piece of porcine muscletissue to simulate a blood vessel as a target for USF imaging. FIG. 8Aschematically illustrates the configuration of the tissue sample, thetube, the excitation light source, the fluorescence collection fiber,and the HIFU transducer used for imaging. The porcine tissue had athickness of approximately 8 mm (in the z-direction of FIG. 8A) and awidth of approximately 20 mm (in the x-direction). The tube was insertedinto the tissue along the y direction. The distance from the tube centerto the top surface of the tissue was approximately 4 mm. A fiber bundlewith a diameter of approximately 3 mm (Edmund Optics NT39-366, NewJersey, USA) was used to deliver the excitation light from a laser tothe bottom of the tissue to excite fluorophores switched to the on stateby exposure to the HIFU beam. A second fiber bundle (Edmund OpticsNT42-345) was placed on the top of the tissue to collect USF photons. A2.5 MHz HIFU transducer (H-108, Sonic Concepts, Washington, USA; activediameter: 60 mm; focal length: 50 mm) was positioned at the bottom ofthe tissue and focused on the tube region. To efficiently transmit theacoustic energy into the tissue, the HIFU transducer, the bottom surfaceof the tissue sample, and the fiber bundle for delivering the excitationlight were submerged in water. For imaging the tube two-dimensionally,the HIFU transducer was scanned or translated in the x-y plane.

USF Imaging System

The setup of the USF imaging system is illustrated schematically in FIG.8B. The system included four primary subsystems: (1) an opticalsubsystem, (2) an ultrasonic subsystem, (3) a temperature measurementsubsystem, and (4) an electronic control subsystem. The opticalsubsystem included components for the delivery of the excitation lightor beam of electromagnetic radiation and the collection of the emissionlight. The excitation light was generated using an 808 nm diode laser(MDL-III-808R) and was delivered to the bottom of the sample tissue viathe fiber bundle described above. A band pass filter F1 (FF01-785/62-25,Semrock, New York; central wavelength: 785 nm; bandwidth: 62 nm) wasused as an excitation filter to clean up any undesirable sidebandcomponents of the diode laser located in the pass band of the emissionfilters. The laser was operated in a continuous wave (CW) mode; however,the sample illumination times and durations were controlled using a fastmechanical shutter (UNIBITZ LS3T2, New York) that was triggered by apulse delay generator (PDG, P400, Highland, California). The shutter hada response time of 0.5 ms. Alternatively, it is also possible to use apulse laser rather than a CW laser. The emitted photons collected viathe second fiber bundle described above were delivered to a set ofemission filters and then received by a photomultiplier tube (PMT). Thecombination of four emission filters permitted maximum rejection of theexcitation photons and passing of the fluorescence emission photons.Specifically, two long pass interference filters (F2 and F5;BLP01-830R-25, Semrock, New York, USA; edge wavelength: 846 nm) and twolong pass absorptive glass filters (F3 and F4; FSR-RG830, Newport,Irvine, Calif., USA, cut-on 830 nm) were positioned as illustrated inFIG. 8B. Two NIR achromatic doublet lenses (AC-254-035-B, Thorlabs, NewJersey, USA) were used to collimate the fluorescence photons for bestrejecting the excitation photons by the interference filters and tofocus the filtered photons onto a cooled and low-noise PMT (H7422P-20driven by a high-voltage source C8137-02, Hamamatsu, Japan). The signalwas further amplified by a low-noise current preamplifier (SR570,Stanford Research Systems, California, USA) and acquired by amultichannel oscilloscope (DPO4102B-L, Tektronix, Oregon, USA).

The ultrasonic subsystem included the HIFU transducer described above,various driving components, an impedance matching network (NWM), aradio-frequency (RF) power amplifier, and a function generator (FG).Specifically, a gated sinusoidal wave signal with a central frequency of2.5 MHz was generated by the FG (33220A, Agilent, California, USA) andwas further amplified by the RF power amplifier (325LA, E&I, New York,USA). The amplified signal was input into the NWM to drive the HIFUtransducer. The HIFU transducer was focused on the silicone sample tube.The HIFU transducer was mounted on a two-dimensional translation stagefor both initial HIFU positioning and subsequent scanning. In theinitial positioning, the HIFU transducer was moved to the position wherethe temperature signal from the thermocouple reached its maximum(indicating that the thermocouple junction was located on the HIFUfocus). This position was considered to be the center of the image. Arectangular area (4.0 mm×1.02 mm) was raster scanned by the HIFUtransducer surrounding the center. The entire ultrasonic subsystem wascontrolled by the PDG, including the firing of the HIFU pulse, thefiring of the excitation light pulse, and the data acquisition of theoscilloscope. The time sequence of these processes is plotted in FIG.8C. In this Example, the ultrasonic exposure time was 300 ms, determinedby the width of the gating pulse from the PDG.

To appropriately synchronize the laser pulse, fluorescence signal, anddata acquisition, the following strategies were adopted. The laser pulsewas delayed approximately 100 ns by coupling the laser beam into a 20 moptical fiber (FT200EMT, Thorlabs Inc., Newton, N.J., USA). When anexcitation light pulse was fired by the laser, a small amount of laserenergy was split by a beam splitter and delivered to a fast photodiode(PD) to generate an electronic pulse. This pulse was used to trigger thePDG. The output of the PDG was used to trigger the oscilloscope for dataacquisition. The triggering time was adjusted by controlling the outputdelay time of the PDG. Thus, the data acquisition of the oscilloscopewas well synchronized and matched with the fluorescence signal. The 100ns delay from the laser pulse was large enough to account for the totalelectronic delay of the trigger signal.

The temperature at the HIFU focus was measured by a micron-sizedthermocouple via an amplifier and a second oscilloscope. Specifically, athermocouple with a small junction size of 75 μm (CHC0003, OmegaEngineering, Connecticut, USA) was disposed in the silicone tube tomeasure HIFU-induced temperature changes. The junction was fixed at thecenter of the scanning area. The output voltage signal from thethermocouple was amplified by an amplifier circuit including ahigh-precision operational amplifier OPA2277 and acquired by anoscilloscope (Infiniium 54830D MSO, Agilent, California, USA). Byscanning the HIFU transducer along the x direction, the temperatureprofile was acquired. The thermocouple signal was found to be linearlyproportional to the temperature, which was previously calibrated outsidethe tissue sample before the test. The measured peak temperature at theHIFU focus was found to be approximately 45° C.

During the ultrasonic exposure period, the tissue temperature at theHIFU focus increased continuously. After the exposure, the temperaturedecreased as a result of thermal diffusion. The excitation lightilluminated the tissue for the final 2 ms right before the end of theultrasonic exposure, and illumination was initiated by opening theshutter. At the same time, the fluorescence signal was acquired by theoscilloscope, which was triggered by a pulse from the PDG. The HIFUtransducer was scanned or translated using a two-dimensional translationstage.

High Resolution USF Images

The HIFU transducer described above was used to ultrasonically image thesample tube in the tissue sample descried above. A pulser/receiver (5073PR, Olympus NDT, USA) was used for both exciting the transducer andreceiving the reflected acoustic echoes. The NWM was also used forimpedance matching between the transducer and the pulser/receiver. Thereflected acoustic signal was amplified by the pulser/receiver andacquired by a digitizer (NI USB 5133) interfaced to a computer. Such areceived signal is usually called an A-line in the ultrasound imagingfield and represents the tissue acoustic impendence distribution alongthe depth (z) direction. One A-line was acquired at each location in thex-y plane. By scanning the HIFU transducer in the x-y plane, a set ofthree-dimensional (x, y, and z) data was acquired. The envelope of eachA-line was calculated for forming the C-mode images at different depths.To compare with the USF image, a set of two-dimensional data in the x-yplane (one of the C-mode images) was extracted by fixing the depth of zat the tube location. The image of FIG. 9B was formed in this manner.

FIG. 9A illustrates a USF image of the tube on the x-y plane. The twovertical dashed lines indicate the locations of the inner edges of thetube. The FWHM and the full-width-at-one-eighth-of-the-maximum (FWEM) ofthe USF image profile along the x direction at each y location werecalculated. The averaged FWHM and FWEM at different y locations were0.48±0.13 mm and 0.68±0.19 mm, respectively. Although the FWHM (0.48 mm)is narrower than the inner diameter of the tube, the FWEM (0.69 mm) isvery close to the tube's inner diameter (0.69 mm). Because the innerdiameter can be considered to be a parameter describing the full size ofthe tube, the FWEM rather than the FWHM can be considered to be aparameter describing the full size of the USF image.

To compare the USF image with a pure ultrasound image, the same samplewas scanned on the x-y plane using the same HIFU transducer via thecommonly used pulse-and-echo method. At each x-y location, the reflectedultrasonic echo from the top inner boundary of the tube was recorded andused to generate the ultrasound image. The result is shown in FIG. 9B.Its averaged FWHM and FWEM were 0.76±0.01 mm and 1.12±0.02 mm,respectively. Both of these values are larger than those of the USFimage. Moreover, if one assumes that the ultrasound speed in muscle isbetween 1,542 and 1,626 m/s, then the theoretical diffraction-limitedlateral focal size (equivalent to the FWHM) of the adopted HIFUtransducer (frequency=2.5 MHz and f-number=0.83) is between 0.512 and0.54 mm, which is also larger than the average FWHM of the USF profilesof the tube. Therefore, methods of imaging described herein can achievea resolution beyond the acoustic diffraction limit.

FIGS. 9C and 9D illustrate comparisons of the intensity profiles ofUSF-generated fluorescence, diffused fluorescence light, ultrasound, andtemperature along the horizontal dashed line marked in FIG. 9A. Inparticular, FIG. 9C illustrates the profiles of the USF signal and thediffused fluorescence signal along the x axis at y=0. FIG. 9Dillustrates the profiles of the USF, ultrasound, and temperature signalsalong the x axis at y=0. Both the USF and ultrasound images werenormalized and interpolated based on a bicubic method. The FWHM of thediffused fluorescence signal was 3.9 mm, which is significantly largerthan the FWHM of the corresponding USF image's profile (0.48 mm) and thetube's inner diameter (0.69 mm). Thus, methods described herein canprovide improved resolution compared to diffused fluorescence methodssuch as fluorescence diffuse optical tomography (FDOT). It should alsobe noted that the temperature profile had a FWHM of 0.66 mm and theultrasound profile had a FWHM of 0.76 mm in FIG. 9D, compared to a USFsignal profile FWHM of 0.54 mm. To acquire the profile of the diffusedfluorescence light, as illustrated in FIG. 9C, the sample was scannedalong the x direction while all the other components remained fixed.Although the HIFU remained off and the temperature was kept at roomtemperature (<LCST), the USF contrast agents still emitted somefluorescence when the laser was on because the USF contrast agents arenot 100 percent off even in the off state. To avoid distortion of theresults by emission filter leakage of the excitation light, a backgroundscan was conducted by filling the tube with water, and this backgrounddata was subtracted from the result acquired from the tube containingthe fluorophores.

Example 3 Ultrasound-Switchable Fluorophores General

A series of ultrasound-switchable fluorophores suitable for use in someembodiments of methods described herein were prepared as follows. Thefluorophores comprised a plurality of FRET donor species and a pluralityof FRET acceptor species either (1) coupled to a linear thermoresponsivepolymer structure, (2) dispersed within a thermoresponsive polymer NPsuch as the NPs described hereinabove in Example 1, (3) coupled to thesurface of a thermoresponsive polymer NP, or (4) partially dispersedwithin and partially coupled to the surface of a thermoresponsivepolymer NP. Structures (1), (3), and (4) are illustrated schematicallyin FIGS. 10-12, respectively. In addition, some properties of variousfluorophores are provided in Tables 2 and 3. Table 3 describesproperties of fluorophores based on linear thermoresponsive polymerstructures. Table 4 describes properties of fluorophores based onthermoresponsive polymer nanoparticles. The nomenclature used in Tables3 and 4 corresponds to the nomenclature described further hereinbelow inthis Example. In addition, measured values reported in Tables 3 and 4were obtained in the manner described in Examples 1 and 2 above.

Structure (1)

In general, thermoresponsive linear polymers were first synthesized andthen fluorescent species were grafted onto the polymer by formingcovalent chemical bonds between appropriate moieties on the polymer andthe fluorescent species, such as hydroxyl, carboxyl, and/or aminemoieties. In some cases, for instance, a carbodiimide coupling schemewas used. Conjugation could also be carried out in other ways. Ingeneral, the donor species had short excitation/emission wavelengths inthe visible region, while the acceptor species had a red/NIR emission(long wavelength). A short wavelength excitation light (for the donor)was used to excite the system, so that little or no acceptor was excitedto a fluorescent state. When a thermoresponsive polymer took on aglobular conformation as described hereinabove, the distance betweendonors and/or acceptors decreased, leading to FRET from the donors tothe acceptors. Therefore, the emission of the acceptor (in longwavelength) could be observed.

To form a series of fluorophores having the general Structure (1), thefollowing materials were used. N-isopropylacrylamide (NIPAM),N-tert-butylacrylamide (TBAm), acrylamide (AAm), acrylic acid (AAc),allylamine (AH), N,N,N′,N′-tetramethyl ethylene diamine (TEMED),ammonium persulfate (APS),N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC),sodium dodecyl sulfate (SDS), N,N′-methylenebisacrylamide (BIS), and7-(2-Aminoethylamino)-N,N-dimethyl-4-benzofurazansulfonamide (DBD-ED)were purchased from Sigma-Aldrich (St. Louis, Mo., USA). SeTau 425mono-N-hydroxysuccinimide (NHS), Square 660 mono-NETS, Seta 700mono-NETS, Seta 633 mono-NETS and Square 660 mono-NH2 were purchasedfrom SETA BioMedicals (Urbana, Ill., USA), and denoted as ST425, Sq660,St700, Sq633, and Sq660a, respectively. All chemicals were used asreceived without further purification.

The thermoresponsive polymer components of the fluorophores having thegeneral Structure (1) included at least three functional portions: (a) aprimary thermoresponsive unit (such as NIPAM); (b) a LCST-controllingunit (such as TBAm or AAm); and (c) a functionalization unit (such asAAc or AH) for coupling to a fluorescent species. Linear polymers weresynthesized through free radical polymerization. All reactions werecarried out in a 250 mL Schlenk tube. The three main steps were asfollows. First, a purging procedure was carried out wherein the reactionmixture was purged with nitrogen for 10 minutes. When an initiator(e.g., APS) or accelerator (e.g., TEMED) was added, oxygen was purged bythree cycles of applying vacuum (1 m) and back-filling with nitrogen (5s). Next, the polymerization reaction was carried out by stirring thereaction mixture under nitrogen for 4 h at room temperature. Finally,the polymer products were purified by dialysis with an appropriatemolecular weight cut-off (MWCO) membrane for three days to removeunreacted monomers, initiator, and other small molecules.

Using P(NIPAM-AAc 200:1) as one example, a general procedure is asfollows. Samples of 1.3644 g NIPAM (monomer) and 4 μL AAc (monomer) at amolar ratio of 200:1 were dissolved in 50 mL deionized (DI) water in theSchlenk tube. During the purging procedure, 0.067 g APS (initiator) and51 μL TEMED (accelerator) were added into the tube. After the reaction,the sample was dialyzed with a 3.5K MWCO membrane. The resultingsolution was collected and freeze-dried for subsequent conjugation withamine-containing fluorescent species. For the conjugation withNETS-containing fluorescent species, the amine-functionalized polymerP(NIPAM-AH) was synthesized using the same protocol as above, exceptusing AH instead of AAc. More generally, the foregoing procedure wasused to synthesize the following thermoresponsive polymers:P(NIPAM-TBAm-AAc 85:15:1), P(NIPAM-TBAm-AAc 185:15:1), P(NIPAM-TBAm-AAc585:15:1), and P(NIPAM-AAm-AAc 200:32:1).

After the synthesis of the polymers, conjugation between the polymersand fluorescent species was carried out using chemical reactions betweencarboxyl and primary amine moieties. In some cases, the fluorescentspecies included NHS, which was reacted with a primary amine of thethermoresponsive polymer (such as an amine present in P(NIPAM-AH)). Inother cases, the fluorescent species included a primary amine that wasconjugated with a carboxyl group of the polymer (such as P(NIPAM-AAc))in the presence of EDC. The conjugation reaction was carried out in a 7mL brown glass tube to protect light-sensitive dyes or fluorescentspecies. The general procedures for conjugation were as follows. Foramine-containing dyes (such as DBD-ED or Sq660a), 5 mg polymer, 25 mgEDC, and 0.3 mg DBD-ED or/and 5 μL Sq660a (stock solution of 1 mg/100 μLdimethyl sulfoxide (DMSO)) were dissolved in 5 mL deionized water in thetube. Then the tube was stirred and reacted overnight at roomtemperature. After completion of the reaction, the conjugates werepurified with appropriate MWCO dialysis membranes as described above.For NHS-containing dyes (such as ST425, St633, Sq660, and St700), 5 mgpolymer and 10 μL dye (stock solution: 1 mg/100 μL DMSO) were dissolvedin 5 mL phosphate buffered saline (PBS, 8 mM sodium phosphate, 2 mMpotassium phosphate, 0.14 M NaCl, 10 mM KCl, pH 8.3-8.6). Then thesolution was stirred and reacted overnight at room temperature. Next, 1mL of 20 mM Tris buffer (pH 7.8) was added to the solution to quench theunreacted NHS moieties of the dye for two hours. Finally, the sample waspurified by dialysis.

It should be noted that, in some cases, DBD-ED, St633, Sq660, and St700were used as polarity-sensitive fluorophores. In other instances, DBD-EDor ST425 was used as a donor species and Sq660(a) was used as anacceptor.

Structure (2)

Fluorophores having the Structure (2) were prepared as described abovein Example 1.

Structure (3)

Fluorophores having the Structure (3) were prepared by first preparingthermoresponsive NPs as described in Example 1 above, except withoutincluding a fluorescent species. Next, fluorescent species were attachedto the surface of the NPs in a manner corresponding to that describedabove for linear thermoresponsive polymers, except 5 mL polymer NPsolution was used rather than 5 mg linear polymer. As one examplefluorophore having the Structure (3), P(NIPAM-AAc 200:1)NPs-DBD-ED-Sq660a was prepared by covalently bonding twoamine-containing dyes (DBD-ED and Sq660a) to the surface of the polymerNPs (P(NIPAM-AAc 200:1) NPs) through carboxyl moieties provided by theAAc monomer.

Structure (4)

Fluorophores having the Structure (4) were prepared by first preparingthermoresponsive NPs as described in Example 1 above and thenconjugating fluorescent species to the surface of the NPs as describedabove for Structure (3). As one example fluorophore having the Structure(4), DBD-ED was encapsulated inside P(NIPAM-AH 86:14) NPs and Sq660 wasattached to the surface of the NPS via conjugation of NHS moieties (fromthe dye) and amine moieties (from the AH monomer). Such fluorophores aredenoted using the general nomenclature DBD-ED@P(NIPAM-AH 86:14)NPs-Sq660, where the species preceding the symbol “@” is encapsulated inthe identified polymer NPs, and the species following the hyphen “-” isconjugated to the surface of the NPs.

TABLE 3 τ_(On)/τ_(off) λ_(ex) & λ_(em) & τ_(On) T_(th) T_(BW)Fluorophore (nm) I_(On)/I_(Off) (ns) (° C.) (° C.) DBD PNIPAM (chain),co-polymerization 470 & 580   4.2 3.5 & 14  31 1 (Donor) P(NIPAM-AAc100:1), post-labeling 470 & 560lp  1.4 4.7 & 4.8 35 8 P(NIPAM-AAc200:1), post-labeling 470 & 560lp  1.6 3.1 & 5.2 36 5 P(NIPAM-AAc600:1), post-labeling 470 & 560lp  1.6 1.9 & 2.5 32 5 P(NIPAM-TBAm-AAc185:15:1), 470 & 560lp  1.8 5.4 & 10  26 4 post-labeling P(NIPAM-AAm-AAc200:32:1), 470 & 560lp  1.1  2 & 2.2 42 9 post-labeling Red dyesP(NIPAM-AH 200:1), post-labeling, 609 & 650/60 4.2 1.1 & 0.9 32 5(acceptor) St633 P(NIPAM-AH 200:1), post-labeling, 609 & 711/25 1.6 2.2& 2.1 35 3 Sq660 P(NIPAM-AH 200:1), post-labeling, 609 & 711/25 0.6 0.7& 1.1 33 8 St700 FRET P(NIPAM- AAc 200:1)-DBD- 470 & 711/25 3.8 3.4 &5.3 34 3 ED, -Sq660a, post-labeling P(NIPAM-TBAm-AAc 470 & 711/25 3 1.7& 5.3 26 3 185:15:1), -DBD- ED, -Sq660a, post-labeling

TABLE 4 τ_(On)/τ_(Off) λ_(ex) & λ_(em) & τ_(On) T_(th) T_(BW)Fluorophore (nm) I_(On)/I_(Off) (ns) (° C.) (° C.) DBD @PNIPAM NPs,encapsulated 470 & 560lp  4 3.3 & 6  35 5 (donor) @P(NIPAM-AAm 86:14)NPs, 470 & 560lp  3.5 2.2 & 3.8 42 9 encapsulated @P(NIPAM-TBAm 185:15)NPs, 470& 560lp  3.7 3.6 & 7.2 31 5 encapsulated @P(NIP AM-AH 86:14)NPs, 470 & 560lp  3 2.6 & 5.3 33 8 encapsulated Red dyes @PNIPAM NPs,encapsulated, 630 & 711/25 0.7 0.7 & 1.2 36 9 (acceptor) St700 @PNIPAMNPs, encapsulated, 609 & 711/25 3.3 1.3 & 2.9 35 5 Sq660 FRET DBD-ED@P(NIPAM-AH 86:14) 470 & 711/25 6.9  1.4 & 3.42 35 7 NPs-Sq660P(NIPAM-AAc 200:1) NPs- 470 & 711/25 5.3 3.3 & 6  35 5 DBD -ED-Sq660aP(NIPAM-TBAm-AAc 185:15:1) 470 & 711/25 6.5 2.7 & 5.2 33 9NPs-DBD-ED-Sq660a P(NIPAM-AAc 200:1) NPs- 456 & 711/25 7  1.5 & 3.65 364 ST425 -Sq660a

Example 4 Ultrasound-Switchable Fluorophores General

A series of ultrasound-switchable fluorophores suitable for use in someembodiments of methods described herein were prepared as follows. Thefluorophores comprised a thermoresponsive polymer and a fluorescentmaterial with an emission peak in the red/NIR portion of theelectromagnetic spectrum. In particular, ADPDI cyanocinnamic acid dye(ADPDICA) was used. The structure of ADPDICA is illustrated in FIG. 13.Some fluorophores were of the general Structure (1) from Example 3hereinabove; other fluorophores were of the general Structure (2) fromExample 3; and still other fluorophores were of the general Structure(3) from Example 3.

Materials

N-isopropylacrylamide (NIPAM), acrylamide (AAm), ammonium persulfate(APS), sodium dodecyl sulfate (SDS), N,N,N′,N′-tetramethyl ethylenediamine (TEMED), N,N′-methylenebisacrylamide (BIS), acrylic acid (AAc),N-tert-butylacrylamide (TBAm), and sodium ascorbate were purchased fromSigma-Aldrich (St. Louis, Mo., USA). All chemicals are used as receivedwithout further purification.

Synthesis

ADPDICA was prepared according to the azadipyrromethene synthesisprocedures described in Bandi et al., “Excitation-Wavelength-Dependent,Ultrafast Photoinduced Electron Transfer inBisferrocene/BF2-Chelated-Azadipyrromethene/Fullerene Tetrads,” Chem.Eur. J. 2013, 19, 7221-7230, and Bandi et al., “Self-Assembled viaMetal-Ligand Coordination AzaBODIPY-Zinc Phthalocyanine andAzaBODIPY-Zinc Naphthalocyanine Conjugates: Synthesis, Structure, andPhotoinduced Electron Transfer,” J. Phys. Chem. C 2013, 117, 5638-5649.Briefly, 3-(4-hydroxyphenyl)-1-phenylprop-2-en-1-one was first preparedby reacting the corresponding 4-hydroxybenzaldehyde, acetophenone, andpotassium hydroxide. This species was subsequently reacted withnitromethane and diethylamine in dry ethanol to obtain3-(4-hydroxyphenyl)-4-nitro-1-phenylbutan-1-one. Next,4-{2-[3-(4-hydroxyphenyl)-5-phenyl-1H-pyrrolylimino]-5-phenyl-2H-pyrrol-3-yl}phenolwas synthesized by reaction with ammonium acetate in ethanol. Then,BF2-chelated4-{2-[3-(4-hydroxyphenyl)-5-phenyl-1H-pyrrol-2ylimino]-5-phenyl-2H-pyrrol-3-yl}phenolwas formed from this product by treating the product withdiisopropylethylamine and boron trifluoride diethyl etherate in dryCH₂Cl₂. The BF2-chelated species was then reacted with the appropriatebenzoic acid in the presence of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) followed bychromatographic purification.

Fluorophores having the general Structure (1) were prepared according tothe protocol described for Structure (1) in Example 3 above, exceptADPDICA was used as the fluorescent species. In addition, purificationwas carried out by dialysis for 3 days using a 3.5 kDa MWCO membrane.Thermoresponsive polymers included P(NIPAM-AH 200:1), P(NIPAM-TBAm-AH185:15:1), and P(NIPAM-AAm-AH 172:28:1). Therefore, the followingfluorophores of general Structure (1) were formed: (a) P(NIPAM-AH200:1)-ADPDICA, (b) P(NIPAM-TBAm-AH 185:15:1)-ADPDICA, and (c)P(NIPAM-AAm-AH 172:28:1)-ADPDICA. The conjugation of ADPDICA to thethermoresponsive polymers was carried out by forming an amide bondbetween the carboxyl groups of ADPDICA and the amine groups provided bythe AH repeating units of the polymer.

Fluorophores having the general Structure (2) were prepared according tothe protocol described for Structure (2) in Example 3 above. Inparticular, the protocol corresponded to that for the DBD-Sq660 FRETsystem. One exemplary fluorophore formed in this manner wasADPDICA@P(NIPAM-AH 200:1) NPs, in which ADPDICA was encapsulated insidethe NPs.

Fluorophores having the general Structure (3) were prepared according tothe protocol described for Structure (3) in Example 3 above. Inparticular, the protocol corresponded to that for the DBD-Sq660 FRETsystem. One exemplary fluorophore formed in this manner was P(NIPAM-AH200:1) NPs-ADPDICA, in which ADPDICA was attached to the surface of theNPs. As with Structure (1), the conjugation of ADPDICA to thethermoresponsive polymer was carried out by forming an amide bondbetween the carboxyl groups of ADPDICA and the amine groups provided bythe AH repeating units of the polymer.

Fluorescence Imaging

The temperature-dependent fluorescence properties of the fluorophoresdescribed above were evaluated at two different excitation wavelengths(λ_(ex)=609 nm or 655 nm) and using a 711 nm/25 nm band pass emissionfilter. The temperature of the fluorophores was controlled by disposingthe fluorophores in a temperature-controlled water bath. The results areprovided in Tables 5 and 6. Table 5 provides data for λ_(ex)=609 nm.Table 6 provides data for λ_(ex)=655 nm. The data in Tables 5 and 6labeled as “λ_(ex)” refers to the emission filters used, where “Ip”refers to a long pass filter.

TABLE 5 λ_(ex), λ_(em) I_(On)/ T_(th) T_(BW) Fluorophore (nm) I_(Off) (°C.) (° C.) P(NIPAM-AH 200:1)-ADPDICA 609, 75.45 33 7 711/25P(NIPAM-TBAm-AH 185:15:1)- 609, 93.59 30 7 ADPDICA 711/25 P(NIPAM-AAm-AH172:28:1)- 609, 188.94 42 7 ADPDICA 711/25 ADPDICA@P(NIPAM-AH 200:1) NPs609, 2.14 35 2 711/25 P(NIPAM-AH 200:1)-NPs-ADPDICA 609, 20.12 34 6711/25

TABLE 6 λ_(ex), λ_(em) I_(On)/ T_(th) T_(BW) Fluorophore (nm) I_(Off) (°C.) (° C.) P(NIPAM-AH 200:1)-ADPDICA 655, 319.95 33 7 711/25P(NIPAM-TBAm-AH 185:15:1)- 655, 417.2 30 7 ADPDICA 711/25 P(NIPAM-AAm-AH172:28:1)- 655, 274.46 42 7 ADPDICA 711/25 ADPDICA@P(NIPAM-AH 200:1) NPs655, 1.79 35 2 711/25 P(NIPAM-AH 200:1)-NPs-ADPDICA 655, 43.38 34 6711/25

Example 5 Methods of Imaging Using Microbubbles

Methods of imaging according to some embodiments described herein werecarried out as follows. First, a series of fluorophores comprisingmicrobubbles was prepared. In one case, the fluorophores includedTargestar-B microbubbles having a plurality of FRET acceptor species (or“fluorophore” species, abbreviated as “F”) and a plurality of FRET donorspecies (or “quencher” species, abbreviated as “Q”) attached to theexterior surface of the microbubbles. Specifically, Alexa Fluor (AF) 546(donor or F) and AF 647 (acceptor or Q) were labeled on the microbubblesurface via biotin-streptavidin coupling.

For imaging, the intensity of the excitation light (532 nm) wasmodulated at 15 MHz. Individual F-Q microbubbles were flowed slowlythrough an ultrasonically and optically transparent microtube. Anultrasound burst with three cycles at a central frequency of 2.25 MHzwas used to expand the F-Q microbubbles. Initially, the fluorescencesignal from the AF 546 was weak due to FRET energy transfer from AF 546to AF 647. However, the fluorescence emission from AF 546 could beultrasonically switched on (with an I_(On)/I_(Off) ratio of about 5 anda τ_(On)/τ_(Off) ratio of about 5) due to the expansion of themicrobubbles during the negative pressure cycles formed by theultrasound bursts. The emission from the acceptor (AF 647) displayed acomplementary behavior.

It was discovered that a well-confined ultrasonic negative pressurefield could be formed using two diffraction-limited ultrasound beams forF-Q-microbubble-based USF imaging. The ultrasound beams were provided bytwo focused 5 MHz transducers. The FWHM of the lateral size of thefocused negative pressure region of each beam was about 450 μm. Thissize was determined primarily by the diffraction and numerical aperture(NA) of the transducer. The axial size of the focus was about 380 μm.This size was primarily determined by the ultrasound pulse length andfrequency and the transducer (assuming the axial resolution is at leasthalf of the pulse width multiplied the ultrasound speed). When the twoultrasound pulses perpendicularly propagated and crossed each other atthe common focal zones, an interfered pressure field was obtained bysumming the two individual, perpendicular fields. In this manner, asmall negative pressure region was formed, wherein the small region hada size smaller than the focal zone of either beam used to form theregion. The FWHM of the main negative pressure region (MNPR) formed bythe two beams was about 165 μm in the lateral direction, which was about2.7 times and 2.3 times narrower than the lateral and axial resolutionsof the individual ultrasound pulses, respectively. A similar result wasfound in the axial direction of the interfered field.

While the MNPR was spatially confined, it was also temporally limiteddue to the propagation of the ultrasound pulses. The lifetime of theconfined MNPR was approximately 0.08-24 μs. This time period was longenough to permit illumination of surrounding tissue using ps lightpulses to excite the fluorophores in the on state. It was also muchlonger than the width of the excitation light pulses, which may bewidened to 1-3 ns in deep tissue due to light scattering in the tissue.Therefore, if needed, multiple light pulses could be provided in asingle time window. By optically illuminating the tissue only withinthis time window (i.e., temporally confining the excitation), it waspossible to avoid background fluorescence noise generated byfluorophores unexpectedly switched-on by the individual ultrasoundpulses before and after the formation of the MNPR. Thus, the USFfluorescence signal could be detected only when both optical andultrasonic pulses were spatially and temporally overlapped.

In addition, it was discovered that the spatial resolution of imagingmethods described herein, in some embodiments, could be further improvedby appropriately selecting the pressure threshold to oscillate amicrobubble described herein. For example, assuming a negative pressurethreshold of 100 kPa and a negative peak pressure in each individualultrasound pulse below this threshold, the fluorophores would not beswitched on until the two ultrasonic pulses are overlapped and the MNPRis formed. When two ultrasonic pulses described above are used to formthe MNPR, the maximum negative pressure of the interference field isdoubled (200 kPa) due to constructive interference. Only the F-Qmicrobubbles within the region where the pressure is above 100 kPa canbe switched on. The full size of this region is about 165 μm and itsFWHM (the spatial resolution) is about 83 μm. However, even smalleractivation region sizes and even high resolution powers can be obtainedby further adjusting the relationship between the switching thresholdvalue (e.g., 100 kPa) and the peak negative pressure provided by eachindividual ultrasonic beam. When the negative pressure switchingthreshold increases (becomes more negative), the size of the region inwhich F-Q microbubbles can be switched on decreases, and therefore thespatial resolution is improved. FIG. 14 illustrates the relationshipbetween the FWHM of the activation region in which F-Q microbubbles areswitched on and the threshold value of the microbubbles, wherein theswitching threshold is represented as a percentage of the maximumnegative pressure of an individual ultrasound beam. When the thresholdis above about 70 percent of the maximum negative pressure, theresolution is quickly improved for both 5 and 10 MHz ultrasoundfrequencies. For example, when the threshold is 90 percent of themaximum negative pressure, the spatial resolution can reach 14 μm and 35μm for the 10 MHz and 5 MHz ultrasound frequencies, respectively, whichis significantly improved compared to the pure ultrasonic lateral andaxial resolutions (450 μm and 380 μm).

In general, improvement of spatial resolution can be accompanied by thedegradation of signal-to-noise ratio (SNR). This degradation can be dueto the smaller volume of the activation region. At a given concentrationof fluorophores, fewer fluorophores may be likely to be found in asmaller volume of a biological environment. As a result, fewerfluorophores may be available in an on state to be excited and a weakerUSF signal may be expected. The extreme, non-zero case occurs when onlya single F-Q microbubble is located in an image voxel (such as 30 μm×30μm×30 μm voxel). Typically, a 2-μm-diameter microbubble can be labeledwith 5×10⁴ molecules/μm², based on the volume of the microbubble, whichis equivalent to a volume concentration of 249 μM, using the bubble'svolume to calculate the concentration. If the volume is taken to be a 30μm×30 μm×30 μm voxel, then this labeling amount is equivalent to avolume concentration of 36 nM. These concentrations are far above thedetection limits (fM-nM) of most optical techniques for tissue imaging.Therefore, using a highly sensitive optical detection system (such as atime-gated and/or photon counting system) can substantially compensatefor any loss in SNR due to the small activation region volume, and it ispossible to detect a single F-Q microbubble in tissue.

Example 6 Ultrasound-Switchable Fluorophores

Additional ultrasound-switchable fluorophores suitable for use inmethods according to some embodiments described herein include thefollowing.

F-Q-HJ Microbubbles

To implement FRET between a donor-acceptor pair on a microbubble'ssurface, a donor and an acceptor can be labeled on a microbubble via aHolliday junction (HJ). FIG. 15 illustrates such a microbubbleschematically. The two crossed lines represent two arms of the HJ, whichis composed of four DNA double helices in the form of a four-wayjunction. The two squares indicate a pair of donor and acceptor speciesthat are labeled at the two ends of the two arms (viastreptavidin-biotin coupling or through the reaction between the nucleicacids of the HJ and an NHS ester attached to the donor and acceptor).The horizontally oriented line indicates the shell of the microbubble onwhich the other two ends of the HJ are attached (via biotin-streptavidincoupling or another coupling scheme). When the bubble is expanded(compressed), the distance Rs is increased (decreased). This results inthe increase (decrease) of the distance between the donor and theacceptor (R_(DA)) with a magnification of (I₁/I₂) and concomitantswitching on (off) of the donor. The initial angle (θ) between the twoarms can be controlled. Due to the relatively large surface area of amicrobubble, numerous F-Q-HJs may be labeled on a single microbubblewithout significant interference. Such a design can narrow the USFtransition band and improve ultrasound-switching efficiency and SNR.

F-Q-Hairpin-NP Microbubbles

Another labeling strategy is to attach a donor-acceptor pair on a DNAhairpin complex (see FIGS. 16 and 17). One end of the hairpin complex isattached to a microbubble surface via biotin-streptavidin coupling, andthe other end is attached to a much smaller gold nanoparticle (Au—NP,tens of nm in diameter) via digoxigenin-antidigoxigenin coupling. TheF-Q (or D-A) labeled DNA hairpin complex consists of three majorcomponents: (1) a hairpin molecule (dotted region in FIG. 16), (2) anoligonucleotide attached to a donor and a digoxigenin, and (3) anoligonucleotide attached to an acceptor and a biotin. Not intending tobe bound by theory, the principle to switch on the donor can bedescribed as follows. An ultrasound pressure wave can accelerate themicrobubble wall and therefore accelerate the Au nanoparticle bystretching the hairpin molecule. The accelerated Au—NP applies anopposite force on the hairpin molecule. When this force is large enough,the hairpin loop can be opened, thereby increasing the donor-acceptordistance and switching on the donor. A force of about 18 picoNewton (pN)can be used to open a hairpin and turn on the donor emission. When theforce is reduced to less than about 6 pN, the hairpin is closed and thedonor is switched off. It is estimated that an ultrasound pulse with a150 kPa pressure wave applied to a microbubble with a diameter of 2 μmand attached to an Au—NP with a diameter of 20 nm can generate a forceof about 20 pN to stretch the hairpin molecules. Therefore, it ispossible to ultrasonically switch on the fluorescence.

F-Q-Hairpin Microbubbles

It is also possible to replace the Holliday Junction of the F-Q-HJmicrobubbles above with a DNA hairpin molecule described above. Its twoends can be annealed to two complementary oligonucleotides. Oneoligonucleotide is labeled with a donor (such as AF 610) and a biotin.Similarly, the other oligonucleotide is attached to an acceptor (such asAF 647) and a biotin. The biotin ends can be attached to astreptavidin-labeled microbubble. When the length of the DNA molecule isshorter than its persistent length (usually about 50 nm), the DNAmolecule behaves like an elastic rod. Therefore, the two arms arenaturally stretched and attached to the microbubble surface. Whenexposure to an ultrasound beam expands the microbubble during a negativepressure cycle, a force is applied on the two ends of the hairpin arms,which can open the hairpin and switch on the donor as described above.

F-Q-DNA-NP Microbubbles

It is also possible to attach a microbubble with relatively smallnanoparticles (tens of nm) via fluorescence-labeled double stranded (ds)DNA molecules. The ds-DNA is attached to the microbubble surface viabiotin-straptavidin coupling. The other end of the ds-DNA is attached toa gold nanoparticle (Au NP) via a thiol linkage. Normally the ds-DNA isbent and flatly absorbed to the surface of the AuNP due to electrostaticattraction, hydrophobic interactions, and ion-dipole dispersiveinteractions between the ds-DNA and the AuNP. Due to the attractionbetween the Au NP and the DNA molecule, the Au NP is close to thefluorescent species that are labeled on one end of the ds-DNA. Thesurface of the AuNP can quench the fluorescent species within arelatively long distance (about 3-20 nm). When an ultrasound pressurewave is applied to compress the microbubble, the accelerated microbubblewall will accelerate the Au NP by stretching the ds-DNA molecule. Whenthe acceleration of the microbubble wall is sufficiently large(controlled by ultrasound pressure strength) that the forces generatedby the electrostatic attraction and other interactions between the DNAand the Au NP, the Au NP cannot experience the same acceleration (due tothe mass of the AuNP), resulting in separation of the fluorescentspecies from the Au NP surface and removal of the quenching effect.

QD-Thermoresponsive Polymer-Acceptor Fluorophores

Another FRET-based fluorophore uses a semiconductor quantum dot such asa CdSe quantum dot as a donor and a small molecule dye as an acceptor.The quantum dot is attached to one or more acceptor dyes using one ormore linkers formed from a thermoresponsive polymer. For example, ared-emitting quantum dot (Qdot® 655, Invitrogen, Inc.) is selected as adonor and a NIR dye (Alexa Fluor 750, Invitrogen, Inc.) as the acceptor.Multiple acceptors (AF 750) are attached on a single donor (Qdot® 655)via thermoresponsive polymers using coupling schemes describedhereinabove. The QD donor has a very long lifetime (approximately 30 ns)and the acceptor AF 750 has a very short lifetime (approximately 0.7ns). When T<LCST, the thermoresponsive polymer exhibits an extended coilor chain conformation, which is relatively long. Therefore, thedistances between the donor and acceptors are generally longer than theFRET quenching range (>40 nm). When a HIFU transducer heats thethermoresponsive polymer above its LCST in a manner describedhereinabove, the polymer makes a transition to a globular conformation,thereby reducing the donor-acceptor distances (<20 nm). As a result,FRET energy transfer occurs. Accordingly, part of the excitation energyof the donor (Qdot® 655) is transferred to the acceptors (AF 750), whichemit photons at NIR wavelengths. These FRET-related photons can have alifetime close to the longer of the donor lifetime and the acceptorlifetime, which is approximately 30 ns in this case. Thus, the emittedNIR photons can be readily detected with high SNR using a time-gateddetection technique described herein. A long pass optical filter can beused to eliminate detection of the QD emission.

Example 7 Ultrasound-Switchable Fluorophores General

A series of ultrasound-switchable fluorophores suitable for use in someembodiments of methods described herein were prepared as follows. Zincphthalocyanine (ZnPC) derivatives were encapsulated into Pluronic F-98micelles or P(NIPAM-TBAm) nanoparticles (NPs) as the contrast agent formethods of imaging according to some embodiments described herein.

Materials

Zinc phthalocyanine (ZnPC), zinc2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (ZnPCTTB), zinc1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine (ZnPCOB), zinc1,2,3,4,8,9,10,11,15,16,17,18,22,23,24,25-hexadecafluoro-29H,31H-phthalocyanine(ZnPCHF), zinc2,3,9,10,16,17,23,24-octakis(octyloxy)-29H,31H-phthalocyanine (ZnPCOO),and zinc 2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine (ZnTTBNPC)were purchased from Sigma-Aldrich (St. Louis, Mo., USA). The chemicalstructures of these ZnPC derivatives are shown in FIG. 18A and FIG. 18B.Tetrabutylammonium iodide (TBAI) (Sigma-Aldrich), Pluronic F-98 (BASF,Florham Park, N.J., USA), NIPAM, TBAm, BIS, 4,4′-Azobis(4-cyanovalericacid) (ACA), and SDS were also used.

Synthesis of ZnPC-Encapsulated Pluronic F-98 Micelle

ZnPC was dissolved in chloroform with the addition of tetrabutylammoniumiodide under sonication for 30 minutes. Meanwhile, Pluronic F-98 wasdissolved in de-ionized (DI) water (pH 8.5) at a concentration of 50mg/mL. The ZnPC chloroform solution was drop-wise added into thePluronic F-98 aqueous solution under vigorous stirring (500 rpm). Theresulting emulsion was further dispersed with a sonication probe(Qsonica) at 40 W for 4 minutes (30 second pulse after every 1 minuterun). The chloroform was evaporated in a fume hood overnight. A clearsolution was obtained which was subsequently filtered using a 0.45 μmmembrane.

Synthesis of ZnPC-Encapsulated P(NIPAM-TBAm) NPs

A mixture of 1.3644 g NIPAM, 0.1247 g TBAm, 0.0131 g BIS, 0.070 g ACAand 0.0219 g SDS were dissolved with 50 mL of DI water (pH 10.5) in a250 mL Schlenk tube, followed by nitrogen purging for 10 minutes. ZnPCDMSO solution (2 mL) was added to the tube, which was then placed undervacuumed and subsequently purged with nitrogen. The pump/purge procedurewas repeated three times in order to provide a nitrogen atmosphereinside the tube. The reaction was carried out at 70° C. overnight. Thereaction was then stopped by loosening the valve to expose theenvironment to air. The sample was dialyzed against DI water using a10-kDa molecular weight cut-off membrane for 3 days to remove excesssurfactants and unreacted material.

Fluorescent Response of ZnPC-Encapsulated Pluronic F-98 Micelles

The temperature-dependent fluorescence properties of the fluorophoresdescribed above were evaluated at an excitation wavelength of λ_(ex)=609nm and using a 711 nm/25 nm band pass emission filter (with theexception of ZnTTBPC, which has a longer emission peak wavelength, forwhich a band pass filter of 765 nm/62 nm was used). The results areprovided in Table 7 and FIGS. 19A-19F.

TABLE 7 λ_(ex), λ_(em) I_(On)/ T_(On)/ T_(th) T_(BW) Fluorophore (nm)I_(Off) T_(Off) (° C.) (° C.) ZnPC 609, 711/25 313.94 1.54 20.3 11.9ZnPC(TTB) 609, 711/25 1450.50 1.87 16.3 11.2 ZnPC(OB)* 609, 711/25 1.32*<1*    * * ZnPC(HF) 609, 711/25 2.71 4.24 28.1 10.2 ZnPC(OO) 609, 711/2535.80 2.02 23.1 11.6 ZnNPC(TTB) 609, 785/62 13.01 1.79 258    9.4 *Notstable.

Fluorescent Response of ZnPC-Encapsulated P(NIPAM-TBAm) NPs

The fluorescence intensity of ZnPC-encapsulated P(NIPAM-TBAm) NPs isshown in FIG. 20A and FIG. 20B. The I_(On)/I_(Off) ratio wasapproximately 1.8 for ZnPC- and P(NIPAM-TBAm)-containing NPs. Thefluorescence lifetime of dye (A) encapsulated inside the NPs in the offstate (T<LCST) was found to be approximately 3 ns (FIG. 20A)

Example 8 Ultrasound-Switchable Fluorophores General

A series of ultrasound-switchable fluorophores according to someembodiments described herein were prepared as follows. Specifically, twoclasses of NIR dyes were used to prepare fluorophores: aza-BODIPYderivatives and zinc phthalocyanine (ZnPC) derivatives. Thermo-sensitivepolymers of Pluronic F-127, Pluronic F-98, and their copolymers with PEGwere used to synthesize nanocapsules for the dyes having differentswitching-on thresholds. The diameters of the nanocapsules were betweenabout 20 nm and about 70 nm, based on measurement by transmissionelectron microscopy (TEM). From the two dye classes above, ADPDICA andZnPC(TTB) were used to form fluorophores. The switching properties ofthese fluorophores are summarized in Table 8.

Materials

ADPDICA was synthesized according to the techniques described in Bandiet al. consistent with Example 4 hereinabove. The chemical structure ofADPDICA is provided in FIG. 13. ZnPC(TTB) was purchased fromSigma-Aldrich (St. Louis, Mo., USA). The chemical structure of ZnPC(TTB)is provided in FIG. 18A. Pluronic F-127 and F-98 were obtained from BASF(Florham Park, N.J., USA). Methoxyl PEG carboxylic acid products(MW=20,000, 30,000, and 40,000 g/mol) were purchased from Nanocs Inc.(New York, N.Y., USA). All chemicals were used directly without furtherpurification. TBAI was also used.

Synthesis

Pluronic F-127 or F-98 (depending on the desired preparation) wasdissolved in DI water (pH 8). The dye/TBAI (molar ratio=1/6) weredissolved in chloroform, and kept in sonication for 30 min. The dye/TBAIchloroform solution was added drop-wise into the Pluronic aqueoussolution while the solution was stirred. The solution was furtherdispersed with a sonicator (Qsonica, LLC., Newtown, Conn., USA) operatedat 20 W for 4 minutes, and the resulting solution was kept stirringunder a fume hood until the chloroform was completely evaporated. Theclear solution that resulted was filtered through a 1.2 μm membrane(Fisher Scientific, Pittsburgh, Pa., USA), and an Amicon Ultracentrifugal filter (10,000 molecular weight cut-off, Millipore,Billerica, Mass., USA).

Characterization of Fluorophores

The optical switching properties of the fluorophores were measuredaccording to the system described hereinabove in Examples 1 and 2. Theresults are provided in Table 8.

TABLE 8 λ_(ex), λ_(em) I_(On)/ T_(th) T_(BW) Fluorophore+ Micelle (nm)I_(Off) (° C.) (° C.) ADPDICA F-127 655, 711/25 230.4 18.5 7.6 F-98 655,711/25 268.6 30.5 4.9 F-98-PEG20K 655, 711/25 272.1 35.5 14.7F-98-PEG30K 655, 711/25 203.8 40.7 15.0 F-98-PEG40K 655, 711/25 214.835.6 15.1 ZnPC(TTB) F-127 655, 711/25 209 18.5 7.6

Example 9 USF Imaging Systems

USF imaging systems suitable for use in some embodiments of methods ofimaging described herein are provided. Specifically, the following threeUSF imaging systems have been tested: (1) a continuous wave (CW) modesystem, (2) a frequency-domain (FD) mode system, and (3) time-domain(TD) mode system. The system configurations and the time sequences fprthese systems are shown in FIGS. 21A-21G.

With a CW mode system, the imaged environment (such as biologicaltissue) is exposed to the HIFU continuously for a short period of time(on the order of milliseconds). The excitation laser is activated orturned on prior to or during the HIFU exposure, generating a USF signal.The fluorescence intensity of the USF signal is then calculated at eachlocation (see FIG. 21A and FIG. 21B).

With an FD mode system, the HIFU exposure is modulated with apredetermined frequency for a brief period. Moreover, the USF signal hasthe same frequency as the modulated HIFU frequency. By using a lock-inamplifier, the modulated USF signal can be detected with very highsensitivity (FIG. 21C-21E). In addition, the laser illumination can alsobe modulated to a relatively high frequency, such as from kHz to MHz,while the ultrasound signal operates in a CW mode (FIG. 21C-21E). Thus,the lock-in amplifier can be used to detect fluorescence signal changescaused by the ultrasound. Moreover, the use of a lock-in amplifier canpermit high sensitivity of the system to the USF signal.

With a TD mode system, the HIFU exposure is continuous over a short timeperiod (on the order of milliseconds), and the laser illuminates apico-second pulse to excite the USF contrast agents just after HIFUheating has ended. Thus, the emitted USF signal becomes a pulse with awidth on the order of tens of nanoseconds due to the long fluorescencelifetime of the switched-on fluorophores (FIG. 21F and FIG. 21G). Incontrast, background noise decays much more quickly due to the shortfluorescence lifetimes of fluorescent background species. Therefore, byusing a time-gating detection technique, the USF signal can betemporally separated from the background noise by acquisition of justthe tail portion of the signal. Thus, both sensitivity andsignal-to-noise ratio are significantly increased over certain othermethods/systems.

In addition, it should be noted that to avoid significant thermaldiffusion in the heating period, the HIFU exposure time can be limitedto milliseconds, a much shorter duration than the thermal diffusion timeconstant.

Example 10 Method of Imaging Providing an Improved SNR

Methods of imaging according to some embodiments described herein werecarried out as follows. First, a series of ultrasound-switchablefluorophores were prepared according to Example 7 above. Thefluorophores included (1) a fluorophore comprising ADPDICA encapsulatedin nanocapsules or micelles formed from Pluronic F127 polymer and (2) afluorophore comprising ICG encapsulated in PNIPAM nanoparticles. Thesefluorophores were prepared in a manner similar to that described inExamples 7 and 8 above. The peak emission wavelengths of the twodiffering fluorophores were approximately 710 nm and 810 nm,respectively. In addition, the dynamic behaviors of the USF signals ofthese fluorophores were different from each other and were alsodifferent from noise, as illustrated in FIG. 22. Specifically, FIG. 22Ashows a temporal decay profile of the USF signal of theADPDICA-containing fluorophore. FIG. 22B shows the temporal decayprofile of the background or noise signal for the imaging experimentusing the ADPDICA-containing fluorophore. Similarly, FIG. 22E shows atemporal decay profile of the USF signal of the ICG-containingfluorophore. FIG. 22F shows the temporal decay profile of the backgroundor noise signal for the imaging experiment using the ICG-containingfluorophore. Not intending to be bound by theory, it is believed thatthe differing temporal decay profiles of the two fluorophores was due toone or more of (1) the different environmental sensitivities of thefluorescent dyes; (2) the different structures of the fluorophores(e.g., micelle versus nanoparticle); and (3) the differentthermosensitive polymers used to form the fluorophores. The differingemission profiles of the differing ultrasound-switchable fluorophorescan permit multiplexed USF imaging.

Specifically, it should be noted that the emission profiles of thefluorophores shown in FIG. 22A and FIG. 22E have a unique and consistentshape for given experimental conditions, such as the conditionsdescribed above. It should further be noted that once the instrument andexperimental conditions are fixed, the shape of the USF dynamic curvefor a given fluorophore does not change and is independent of the signalstrength. Each signal increases in intensity to a peak value at a fixedtime point and then decays or reduces in intensity. In contrast, thebackground signals (FIG. 22B and FIG. 22F) fluctuate irregularly and donot exhibit any correlation with other noise signals. More importantly,the background signals do not exhibit the same temporal decay profile asthe ultrasound fluorescence signals exhibit. Therefore, the backgroundsignals are not strongly correlated with the ultrasound fluorescencesignals mathematically. Not intending to be bound by theory, it isbelieved that the differing temporal decay profiles allow any USF signalto be selected as a reference signal for carrying out a correlationanalysis of a total photoluminescence signal detected at a specificlocation within an environment. When the correlation is carried out, acorrelation coefficient (CrC) can be calculated. It has been discoveredthat USF-related signals have a large CrC, while the background signalshave a small CrC when such an analysis is performed. Thus, the CrC canbe used to differentiate a USF signal from background noise byappropriately selecting a CrC threshold, as described furtherhereinabove.

In one-color USF imaging, a small silicone tube (inner diameter: 0.31mm; outer diameter: 0.64 mm) was filled with an aqueous solution of afluorophore described above and embedded into a piece of porcine muscletissue. This structure was intended to simulate a blood vessel as thetarget for the USF imaging experiment. The thickness of the tissue wasapproximately 12 mm. The distance from the tube center to the topsurface of the tissue was approximately 6 mm. A focused ultrasound beamwas used to externally and locally switch the fluorophores inside thetube from an off state to an on state, as described further hereinabove.The emitted fluorescence photons were collected by a cooled andlow-noise PMT. After raster scanning the sample with the ultrasoundbeam, USF images were generated in a manner described hereinabove.Specifically, for each of a plurality of locations within the sample, acorrelation analysis was carried out to compare the total detectedphotoluminescence signal to the reference signal. In this case, atypical USF signal was selected from the USF image to serve as areference signal. The correlation coefficient was calculated accordingto Equation (1) above. Because the background signal did not follow theunique dynamic pattern of the reference signal, as shown in FIG. 22, thecorrelation coefficient for background signals was zero, close to zero,or very small (such as <0.3). In this manner, noise could besignificantly suppressed by multiplying the detected signal comprisingthe background signal with the correlation coefficient associated withthe background signal for the relevant location within the environment.

In this manner, the SNR of the USF image was dramatically increased. USFimages before and after correlation processing are shown in FIG. 22 andFIG. 23. Specifically, FIG. 22C/FIG. 23C and FIG. 22D/FIG. 23Dillustrate the spatial fluorescence emission profile of theADPDICA-based fluorophore before and after thecorrelation/multiplication process, respectively. The SNR of those twoprofiles were calculated as 88 and 300, respectively. Similarly, thespatial fluorescence emission profiles of the ICG-based fluorophorebefore and after correlation analysis are illustrated in FIG. 22G/FIG.23A and FIG. 22H/FIG. 23B, respectively. For the ICG-based fluorophore,the SNRs were 31 and 345 before and after correlation, respectively.

Example 11 Method of Multiplexed Imaging

A method of multiplexed imaging according to one embodiment describedherein was carried out as follows. First, ADPDICA-containing andICG-containing ultrasound-switchable fluorophores were prepared asdescribed above in Example 10. Next, the foregoing fluorophores wereimaged using an imaging system similar to that described above inExample 2. The excitation light source was a diode laser with anexcitation wavelength of 671 nm (MLL-FN-671). One 673/11 band-passfilter (central wavelength: 673 nm; bandwidth: 11 nm) was applied as theexcitation filter, and three long-pass filters (edge wavelength: 715 nm)and two long-pass absorptive filters (edge wavelength: 690 nm) were usedas the emission filter.

For two-color USF imaging, a small tubes (inner diameter: 0.31 mm; outerdiameter: 0.64 mm) was embedded into a scattered silicone phantom. Thescattering material was TiO₂. The thickness of the phantom wasapproximately 12 mm. The tube was filled a mixture of 350 μL of anaqueous solution of the ADPDICA-based fluorophore and 250 μL of anaqueous solution of the ICG-based fluorophore. The two solutionscontained the same weight of fluorophore per solution volume. A focusedultrasound beam was used to externally and locally switch on and off thefluorophores inside the tubes. Emitted photons were collected by acooled and low-noise PMT. After raster scanning with the ultrasoundbeam, USF images were obtained in a manner described hereinabove.

Specifically, detected photoluminescence signals were orthogonallydecomposed into first basis vectors corresponding to a normalizedultrasound fluorescence signal of the ADPDICA-based fluorophore andsecond basis vectors corresponding to a normalized ultrasound signal ofthe ICG-based fluorophore. Thus, for a specific location within theenvironment, a total detected photoluminescence signal could berepresented according to Equation (2):

Mixture=a×ADPDICA+b×ICG  (2):

wherein “Mixture” represents the total photoluminescence signal(represented as a vector), “ADPDICA” represents the basis vectorcorresponding to the ADPDICA-based fluorophore, “ICG” represents thebasis vector corresponding to the ICG-based fluorophore, and a and b arethe coefficients for the basis vectors. FIG. 24 illustrates the signalscorresponding to Equation (2) for one specific location. Specifically,FIG. 24A illustrates the component signal of the ADPDICA-basedfluorophore, FIG. 24B illustrates the component signal of the ICG-basedfluorophore, and FIG. 24C illustrates the total detectedphotoluminescence signal. Using a curve fitting algorithm in MATLAB, thebasis vector coefficient a was determined to be 3.9, and the basisvector coefficient b was determined to be 2.6, within 95% confidence forone location within the sample. The ratio of a to b (3.9/2.6=1.5) wasclose to the composition of the mixture (350/250=1.4).

Various embodiments of the present invention have been described infulfillment of the various objectives of the invention. It should berecognized that these embodiments are merely illustrative of theprinciples of the present invention. Numerous modifications andadaptations thereof will be readily apparent to those skilled in the artwithout departing from the spirit and scope of the invention.

That which is claimed is:
 1. A method of imaging comprising: (a)disposing a first ultrasound-switchable fluorophore and a secondultrasound-switchable fluorophore in an environment; (b) exposing theenvironment to an ultrasound beam to create an activation region withinthe environment; (c) disposing the first fluorophore within theactivation region to switch the first fluorophore from an off state toan on state and/or disposing the second fluorophore within theactivation region to switch the second fluorophore from an off state toan on state; (d) exposing the environment to a beam of electromagneticradiation, thereby exciting the first fluorophore and/or the secondfluorophore; (e) detecting a first photoluminescence signal at a firstlocation within the environment, the first photoluminescence signalcomprising at least one of a first ultrasound fluorescence signalemitted by the first fluorophore, a first fluorescence signal emitted bythe second fluorophore, and a first background signal; (f) correlatingthe first photoluminescence signal with a first reference signal togenerate a first correlation coefficient for the first location; and (g)multiplying the first photoluminescence signal by the first correlationcoefficient for the first location to generate a first modifiedphotoluminescence signal for the first location.
 2. The method of claim1, wherein correlating the first photoluminescence signal with the firstreference signal comprises comparing a temporal intensity decay profileof the first photoluminescence signal to a temporal intensity decayprofile of the first reference signal.
 3. The method of claim 2, whereinthe correlation coefficient for the first location is generatedaccording to Equation (1): $\begin{matrix}{{\rho_{I,R} = \frac{( {\sum{{I(t)}{R(t)}}} ) - \frac{( {\sum{I(t)}} )( {\sum{R(t)}} )}{N}}{\sqrt{( {{\sum{I(t)}^{2}} - \frac{( {\sum{I(t)}} )^{2}}{N}} )( {{\sum{R(t)}^{2}} - \frac{( {\sum{R(t)}} )^{2}}{N}} )}}},} & (1)\end{matrix}$ wherein ρ_(I,R) is the correlation coefficient for thefirst location, I(t) is the temporal intensity decay profile of thefirst photoluminescence signal at the first location, R(t) is thetemporal intensity decay profile of the first reference signal, and N isthe number of the time point in I(t) and R(t).
 4. The method of claim 1,wherein the correlation coefficient for the first location is a binnedcorrelation coefficient.
 5. The method of claim 1 further comprising:(f₂) correlating the first photoluminescence signal with a secondreference signal to generate a second correlation coefficient for thefirst location; and (g₂) multiplying the first photoluminescence signalby the second correlation coefficient for the first location to generatea second modified photoluminescence signal for the first location,wherein the first reference signal corresponds to the first ultrasoundfluorescence signal of the first fluorophore, and wherein the secondreference signal corresponds to the first ultrasound fluorescence signalof the second fluorophore.
 6. The method of claim 5 further comprising:(e_(n)) detecting n additional photoluminescence signals at n additionallocations within the environment, the n additional photoluminescencesignals comprising at least one of an nth additional ultrasoundfluorescence signal emitted by the first fluorophore, an nth additionalultrasound fluorescence signal emitted by the second fluorophore, and annth additional background signal; (f_(1n)) correlating the n additionalphotoluminescence signals with the first reference signal to generate nadditional first correlation coefficients for the n additionallocations; (g_(1n)) multiplying the n additional photoluminescencesignals by the n additional first correlation coefficients for the nadditional locations to generate n additional first modifiedphotoluminescence signals for the n additional locations, (f_(2n))correlating the n additional photoluminescence signals with the secondreference signal to generate n additional second correlationcoefficients for the n additional locations; (g_(2n)) multiplying the nadditional photoluminescence signals by the n additional secondcorrelation coefficients for the n additional locations to generate nadditional second modified photoluminescence signals for the nadditional locations, wherein n is an integer between 1 and
 1000. 7. Themethod of claim 6 further comprising: (h₁) combining the first modifiedphotoluminescence signal for the first location and the n additionalfirst modified photoluminescent signals for the n additional locationsto generate a spatial plot of ultrasound fluorescence emitted by thefirst fluorophore within the environment; and (h₂) combining the secondmodified photoluminescence signal for the first location and the nadditional second modified photoluminescent signals for the n additionallocations to generate a spatial plot of ultrasound fluorescence emittedby the second fluorophore within the environment.
 8. A method of imagingcomprising: (a) disposing a first ultrasound-switchable fluorophore anda second ultrasound-switchable fluorophore in an environment; (b)exposing the environment to an ultrasound beam to create an activationregion within the environment; (c) disposing the first fluorophorewithin the activation region to switch the first fluorophore from an offstate to an on state and/or disposing the second fluorophore within theactivation region to switch the second fluorophore from an off state toan on state; (d) exposing the environment to a beam of electromagneticradiation, thereby exciting the first fluorophore and/or the secondfluorophore; (e) detecting a first photoluminescence signal at a firstlocation within the environment, the first photoluminescence signalcomprising at least one of a first ultrasound fluorescence signalemitted by the first fluorophore and a first ultrasound fluorescencesignal emitted by the second fluorophore; and (f) orthogonallydecomposing the first photoluminescence signal into a first basis vectorcorresponding to a normalized ultrasound fluorescence signal of thefirst fluorophore and a second basis vector corresponding to anormalized ultrasound fluorescence signal of the second fluorophore. 9.The method of claim 8 further comprising: (g₁) determining a basisvector coefficient a for the normalized ultrasound fluorescence signalof the first fluorophore at the first location; and (g₂) determining abasis vector coefficient b for the normalized ultrasound fluorescencesignal of the second fluorophore at the first location.
 10. The methodof claim 9 further comprising: (h₁) multiplying the normalizedultrasound fluorescence signal of the first fluorophore by thecoefficient a to generate a separated ultrasound fluorescence signal ofthe first fluorophore at the first location; and (h₂) multiplying thenormalized ultrasound fluorescence signal of the second fluorophore bythe coefficient b to generate a separated ultrasound fluorescence signalof the second fluorophore at the first location.
 11. The method of claim10 further comprising: (e_(n)) detecting n additional photoluminescencesignals at n additional locations within the environment, the nadditional photoluminescence signals comprising at least one of an nthadditional ultrasound fluorescence signal emitted by the firstfluorophore and an nth additional ultrasound fluorescence signal emittedby the second fluorophore; (f_(n)) orthogonally decomposing the nadditional photoluminescence signals into n additional first basisvectors corresponding to a normalized ultrasound fluorescence signal ofthe first fluorophore and n additional second basis vectorscorresponding to a normalized ultrasound signal of the secondfluorophore; (g_(1n)) determining n additional basis vector coefficientsa_(n) for the normalized ultrasound fluorescence signal of the firstfluorophore at the n additional locations; (g_(2n)) determining nadditional basis vector coefficients b_(n) for the normalized ultrasoundfluorescence signal of the second fluorophore at the n additionallocations; (h_(1n)) multiplying the normalized ultrasound fluorescencesignal of the first fluorophore by the n additional coefficients a_(n)to generate n additional separated ultrasound fluorescence signals ofthe first fluorophore at the n additional locations; and (h_(2n))multiplying the normalized ultrasound fluorescence signal of the secondfluorophore by the n additional coefficients b_(n) to generate nadditional separated ultrasound fluorescence signals of the secondfluorophore at the n additional locations, wherein n is an integerbetween 1 and
 1000. 12. The method of claim 11 further comprising: (i₁)combining the separated ultrasound fluorescence signal of the firstfluorophore at the first location with the n additional separatedultrasound fluorescence signals of the first fluorophore at the nadditional locations to generate a spatial plot of ultrasoundfluorescence emitted by the first fluorophore within the environment;and (i₂) combining the separated ultrasound fluorescence signal of thesecond fluorophore at the first location with the n additional separatedultrasound fluorescence signals of the second fluorophore at the nadditional locations to generate a spatial plot of ultrasoundfluorescence emitted by the second fluorophore within the environment.13. The method of claim 10 further comprising: (j₁) correlating theseparated ultrasound fluorescence signal of the first fluorophore with afirst reference signal to generate a first correlation coefficient forthe first reference signal for the first location; (k₁) multiplying theseparated ultrasound fluorescence signal of the first fluorophore by thefirst correlation coefficient for the first reference signal to generatea first modified separated ultrasound fluorescence signal of the firstfluorophore for the first location, (j₂) correlating the separatedultrasound fluorescence signal of the second fluorophore with a secondreference signal to generate a first correlation coefficient for thesecond reference signal for the first location; and (k₂) multiplying theseparated ultrasound fluorescence signal of the second fluorophore bythe first correlation coefficient for the second reference signal togenerate a first modified separated ultrasound fluorescence signal ofthe second fluorophore for the first location, wherein the firstreference signal corresponds to the first ultrasound fluorescence signalof the first fluorophore, and wherein the second reference signalcorresponds to the first ultrasound fluorescence signal of the secondfluorophore.
 14. The method of claim 11 further comprising: (j_(1n))correlating the n additional separated ultrasound fluorescence signalsof the first fluorophore with a first reference signal to generate nadditional correlation coefficients for the first reference signal forthe n additional locations; (k_(1n)) multiplying the n additionalseparated ultrasound fluorescence signals of the first fluorophore bythe n additional correlation coefficients for the first reference signalto generate n additional modified separated ultrasound fluorescencesignals of the first fluorophore for the n additional locations;(j_(2n)) correlating the n additional separated ultrasound fluorescencesignals of the second fluorophore with a second reference signal togenerate n additional correlation coefficients for the second referencesignal for the n additional locations; and (k_(2n)) multiplying the nadditional separated ultrasound fluorescence signals of the secondfluorophore by the n additional correlation coefficients for the secondreference signal to generate n additional modified separated ultrasoundfluorescence signals of the second fluorophore for the n additionallocations, wherein the first reference signal corresponds to the firstultrasound fluorescence signal of the first fluorophore, and wherein thesecond reference signal corresponds to the first ultrasound fluorescencesignal of the second fluorophore.
 15. The method of claim 8, wherein theenvironment is a biological environment, a biological phantom material,or a tissue-mimicking phantom material.